{"paper_id":"13ec8896-6f4d-4271-a6de-27f293ae1e25","body_text":"Dual Role of Entamoeba histolytica KERP2 in Regulating Gene Expression and Modulating Host Cell Function for Intestinal Colonization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dual Role of Entamoeba histolytica KERP2 in Regulating Gene Expression and Modulating Host Cell Function for Intestinal Colonization Tomoyoshi Nozaki, Ruofan Peng, Herbert Santos This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6191032/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Entamoeba histolytica , the protozoan parasite responsible for amoebiasis, deploys a complex array of virulence factors to establish infection and evade host defenses. Here, we identify KERP2 as a dual-function effector that regulates both parasite homeostasis and host cell remodeling. Bioinformatic analyses, cellular localization assays, and functional studies show that KERP2 localizes to the parasite nucleus, associates with chromatin, and modulates transcription, particularly regulating cysteine protease expression and sulfur metabolism. Concurrently, KERP2 is translocated into host epithelial cells, where it manipulates the G1/S transition, interacts with cytoskeletal regulators, and promotes actin remodeling, ultimately compromising epithelial barrier function. Our results elucidate how E. histolytica harnesses KERP2 to coordinate intracellular processes in the parasite while orchestrating pathogenic alterations in host cells. These insights shed light on a broader mechanism by which extracellular pathogens deploy multifunctional effectors to optimize virulence and adapt to diverse host environments, providing a valuable framework for studies on pathogen-host interactions beyond amoebiasis. Biological sciences/Microbiology/Pathogens Biological sciences/Microbiology/Parasitology Entamoeba histolytica KERP2 virulence factor host-pathogen interaction cytoskeletal remodeling cysteine proteases epithelial barrier integrity amoebiasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Pathogens have evolved sophisticated mechanisms to hijack host cellular functions, enabling their survival, replication, and transmission. Unlike commensal microbes, which coexist with the host without triggering strong immune responses, pathogenic microbes actively manipulate host cells to evade immune detection and create environments favorable for infection (Arrieta and Finlay, 2012 ; Littman and Pamer, 2011 ; Tanoue et al., 2010 ). Intracellular pathogens, such as Mycobacterium tuberculosis , Listeria monocytogenes , Plasmodium falciparum , and Toxoplasma gondii , invade host cells to escape immune surveillance and establish specialized intracellular niches (Disson et al., 2021 ; Koch and Mizrahi, 2018 ; Lourido, 2019 ; Maier et al., 2019 ). These pathogens subvert host cellular machinery to facilitate their replication by modifying endocytic trafficking, preventing lysosomal fusion, or hijacking host cytoskeletal components for movement and dissemination. In contrast, extracellular pathogens remain outside host cells but still engage in complex interactions with host tissues to establish infection and evade immune responses. Many extracellular bacteria, such as Vibrio cholerae , Streptococcus pneumoniae , and Staphylococcus aureus , produce virulence factors like toxins or adhesins that modulate host signaling pathways and immune responses (Cho et al., 2021 ; Howden et al., 2023 ; Weiser et al., 2018 ). Similarly, extracellular parasites, including Trypanosoma brucei , Giardia lamblia , and Entamoeba histolytica , have evolved intricate strategies to manipulate host cell functions despite not residing within them (Begum et al., 2021 ; Einarsson et al., 2016 ; Romero-Meza and Mugnier, 2020 ). Extracellular parasites exploit host cell surfaces, secreted factors, and molecular mimicry to ensure their survival and persistence. Some, like T. brucei , evade immune recognition by continuously altering their surface glycoproteins (variant surface glycoproteins, VSGs) through antigenic variation (Mugnier et al., 2015 ; Mugnier et al., 2016 ). Others, such as Giardia , disrupt host epithelial barrier integrity by attaching to the intestinal epithelium via its ventral adhesive disc, a microtubule-based structure (Lanfredi-Rangel et al., 1999 ; Schwartz et al., 2012 ). This attachment induces mechanical stress, disrupting tight junction proteins such as claudin-1, occludin, and ZO-1, ultimately increasing intestinal permeability (Buret et al., 2002 ; Maia-Brigagao et al., 2012 ). Additionally, Giardia also secretes cysteine proteases that degrade host mucins and tight junction proteins, exacerbating epithelial damage and facilitating nutrient uptake (Bhargava et al., 2015 ; Liu et al., 2018 ). Among extracellular protozoan parasites, E. histolytica is particularly notable for its dual role as both a commensal and a pathogen in the human gastrointestinal tract. E. histolytica is responsible for amoebiasis, a disease of significant global health concern that affects up to 50 million people annually, causing over 70,000 deaths worldwide (Bercu et al., 2007 ; Kantor et al., 2018 ; Ximenez et al., 2010 ). Infections often begin asymptomatically but can progress to severe symptoms, including abdominal pain, watery or bloody diarrhea, and, in some cases, life-threatening complications such as liver abscesses, pneumonia, purulent pericarditis, and cerebral amoebiasis (El-Dib, 2017 ; Kantor et al., 2018 ; Sharma and Ahuja, 2003 ). Within the intestine, E. histolytica induces severe inflammation, tissue perforation, and fulminant amoebic colitis, leading to potentially fatal outcomes (Shirley et al., 2018 ; Zulfiqar et al., 2024 ). Although infection rates are declining in many low and middle income countries, an increase has been observed in high-income East Asian regions including Japan and Taiwan and in parts of Europe over the past two decades (Fu et al., 2023 ; Lin et al., 2022 ; Yanagawa et al., 2022 ). Populations at high risk include residents of endemic regions (e.g., Mexico, Central and South America, Asia, Africa, and the Pacific Islands), immigrants and travelers from these areas, men who have sex with men (MSM), and people living or working in group facilities (Ansart et al., 2005 ; Hung et al., 2012 ; Stanley, 2003 ; Weinke et al., 1990 ). The coexistance of asymptomatic carriers and symptomatic individuals highlights E. histolytica’s potential for widespread transmission and underscores the critical need to elucidate its pathogenic mechanisms. Unlike many other extracellular parasites, E. histolytica employs a particularly aggressive mechanism of host interaction known as trogocytosis - a process in which the parasite physically nibbles away portions of host cell membranes, leading to gradual cell destruction (Ralston et al., 2014 ). This mechanism differs from phagocytosis as it occurs while the host cells are still alive, preceding their lysis. Additionally, this parasite deploys a diverse array of virulence factors to adapt to the host environment, evade immune defenses, and establish infection (Stanley, 2003 ). E. histolytica secretes cysteine proteases (EhCPs) that degrade host extracellular matrix components, such as collagen, fibronectin, and laminin, facilitating tissue invasion and causing intestinal ulceration (Li et al., 1995 ; Lidell et al., 2006 ; Moncada et al., 2003 ; Schulte and Scholze, 1989 ). The parasite also releases lectins, such as the Gal/GalNAc lectin, which bind to host glycoproteins and trigger apoptotic and necrotic pathways, further contributing to epithelial damage (Blazquez et al., 2007 ; Huston et al., 2003 ). The pathogenicity of E. histolytica is driven by a complex network of virulence factors, many of which remain poorly characterized (Biller et al., 2014 ; Faust and Guillen, 2012 ). While cysteine proteases such as EhCP-A5 are well-characterized for their roles in disrupting tight junctions, triggering inflammation, and facilitating tissue invasion, the contributions of other effector proteins remain less understood (El-Dib, 2017 ). Recently, the KERP family of lysine- and glutamic acid-rich proteins has emerged as a key player in E. histolytica’s interaction with host cells. The KERP family includes three proteins, KERP1, 2, and 3, characterized by their high lysine and glutamic acid content. Both KERP1 and KERP2, but not KERP3, have been identified in E. histolytica membrane fractions and exhibit the capacity to bind to the brush border of Caco-2 cells, with KERP1 extensively studied as a virulence factor for its role liver abscess formation (Santi-Rocca et al., 2008 ; Seigneur et al., 2005 )​. While KERP1 was found to only be expressed in E. histolytica but not in non-virulent strains such as E. dispar , KERP2 is widely present in Entamoeba species. Current KERP2 genetic studies suggesting its involvement in amoebic liver abscess and asymptomatic infections through polymorphisms in the kerp2 locus​ (Das et al., 2021 ). These findings suggest selective evolutionary pressure on KERP2, pointing to its potential role in parasites adaptation, however, the host-interacting functions and mechanisms of KERP2 remains poorly explored. Our study suggested that KERP2 acts as a critical regulator balancing the virulence of E. histolytica , allowing the host cell cycle to proceed under pressure, and disrupting cytoskeletal dynamics to render the barrier resistance, finally should contribute to the enhancement of parasitic colonization and residence. These insights not only advance our understanding of KERP2’s dual role in E. histolytica’s adaptability and pathogenic potential but also highlight its significance as a versatile virulence factor. Results In Silico Analysis of KERP2 To investigate the evolutionary relationships among KERPs, we compared their primary structures and found that KERP2 (EHI_065630) shares 67.5% sequence identity with KERP3 (EHI_198680), whereas both are distinct from KERP1 (EHI_098210). Using KERP2 as a query, BLASTp and HMMER searches identified regional conservation with DEK-like proteins (Fig. S1 A). To further examine the evolutionary history of KERP2, we performed maximum likelihood phylogenetic reconstruction using IQ-TREE, incorporating homologous sequences from a broad range of eukaryotic taxa (Fig. 1 A). The results revealed that KERP2 and KERP3 are restricted to Entamoeba species, forming a well-supported monophyletic group, suggesting lineage-specific conservation. However, their evolutionary relationship to homologs in fungi, algae, plants, and metazoans is poorly supported, indicative of early divergence from other eukaryotic orthologs (Fig. 1 A). To assess selection pressure acting on KERP2 within Entamoeba species, dN/dS (ω) analysis was performed, yielding a ratio of 0.07979, indicating strong purifying selection. This suggests that KERP2 is functionally constrained within Entamoeba , maintaining its conserved role rather than undergoing rapid adaptation. Notably, when using KERP2 DNA sequences to search within Amoebozoa, two species, Polysphondylium pallidum and Dictyostelium purpureum , showed partial conservation in specific regions (Fig. S1 B). However, these matches were relatively weak, suggesting that KERP2-like sequences may exist as distant remnants in other Amoebozoans but have not been retained as functional orthologs. Intracellular localization of KERP2 in E. histolytica To investigate the intracellular localization of KERP2 in E. histolytica , we generated an amebic line expressing HA-tagged KERP2 and a truncated variant, HA-KERP2 ∆185–239 , which lacks the coiled-coil domain. (Fig. 2 A). Immunofluorescence analysis (IFA) with line intensity profiling revealed distinct localization patterns (Fig. 2 B). Wild-type HA-KERP2 predominantly localized to the nuclear periphery and nucleoplasm, with punctate signals in the cytosol. In contrast, HA-KERP2 ∆185–239 exhibited a significant reduction in nuclear localization and was redistributed to the cytosol. To validate these observations, we performed live-cell imaging using GFP-HA-tagged constructs. The results consistent with the IFA data, with GFP-HA-KERP2 predominantly localized in the nucleus (Videos S1), while GFP-HA-KERP2 ∆185–239 remained cytosolic (Video S2). Immuno-electron microscopy (EM) further demonstrated HA-KERP2 enrichment in electron-dense nuclear regions, typically associated with chromatin-rich areas (Fig. 2 C). Additionally, HA-KERP2 signals were detected in small electron-dense granule (EDG)-like structures, large vesicles, and diffusely distributed in the cytosol (Fig. 2 D). To complement our imaging analyses, we conducted subcellular fractionation to biochemically assess the distribution of KERP2 variants. Cellular fractions were separated into a 14,000 × g pellet (nuclear fraction, containing nuclei, heavy organelles, and membrane components) and a supernatant (cytosolic fraction, containing cytosolic and light vesicular components) (Fig. 2 E-F). HA-KERP2 was predominantly enriched in the nuclear fraction, consistent with its nuclear and membrane association. In contrast, HA-KERP2 ∆185–239 was primarily detected in the cytosolic fraction, further supporting its cytosolic localization and aligning with our imaging results. Cysteine synthase (CS) and histone were used as cytosolic and nuclear markers, respectively. Gene silencing of KERP2 reveals its potential role in regulation of parasitic activities To investigate the functional role of KERP2, we generate a KERP2 -knockdown strain (psAP-KERP2gs), utilizing small interfering RNAs with the psAP-2-Gunma plasmid. Quantitative reverse transcription PCR (qRT-PCR) confirmed a near-complete reduction of KERP2 transcript levels (Fig. 3 A, Fig. S2A). Cell growth assays showed no significant differences between the KERP2gs and psAP-mock strains (Fig. 3 B, Fig. S2B-C). To assess transcriptional changes resulting from KERP2 knockdown, we performed RNA-Seq analysis. Principal component analysis (PCA) showed distinct clustering between KERP2gs and psAP-mock strains (Fig. S2D). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses identified upregulation of genes associated with proteolysis regulation, sulfur amino acid metabolism, and amoebiasis in KERP2gs (Fig. 3 C). Genes showing notable increases in expression included two cysteine synthases (EHI_024230, 7.74-fold; EHI_160930, 4.68-fold) and methionine γ-lyase (EHI_057550, 18.3-fold) (Fig. 3 D). Additionally, cysteine protease (EHI_010850) was upregulated (2.11-fold), along with three pore-forming peptides (EHI_169350, 4.26-fold; EHI_194540, 2.71-fold; EHI_15940, 2.2-fold). To confirm the regulation role of KERP2 in amoebiasis-related genes, we evaluated changes in cysteine protease (CP) activity. Intracellular CP activity across four E. histolytica strains, including HA-KERP2-overexpressing and HA-mock control strains in the G3 background, was measured. KERP2gs strain exhibited a 1.29-fold higher intracellular CP activity (8.05 ± 0.49) compared to psAP-mock (6.25 ± 0.25, p < 0.0001). In contrast, HA-KERP2 overexpression resulted in a 0.89-fold reduction in intracellular CP activity (5.39 ± 0.19) compared to HA-mock (6.08 ± 0.18, p = 0.003) (Fig. 3 E, Fig. S2E-F). Differences were also observed in released CP activity. The KERP2gs strain exhibited a 6.63-fold increase in released CP activity (7.89 ± 0.15) compared to psAP-mock (1.19 ± 0.12, p < 0.0001). In contrast, HA-KERP2 overexpression resulted in lower released CP activity, with HA-KERP2 cells showing 0.75-fold the activity of HA-mock cells (1.39 ± 0.35 vs. 1.85 ± 0.47, p = 0.032) (Fig. 3 F, Fig. S2G-H). Nuclear co-immunoprecipitation revealed KERP2’s role and its trafficking mechanisms in E . histolytica To elucidate KERP2 function through protein-protein interactions, co-immunoprecipitation (co-IP) followed by mass spectrometry (MS) analysis was performed on four biological replicates. HA-KERP2, HA-KERP2 ∆185–239 , and HA-mock samples were fractionated into nuclear and cytosolic fractions before undergoing independent co-IP. Western blotting and Flamingo staining confirmed the success of co-IP (Fig. S3A-D). KERP2 was consistently detected in all HA-KERP2 pull-down samples and identified by MS, except in the nuclear fraction of Trial 3, where non-specific binding in the HA-mock control led to its exclusion from further analysis. Significant KERP2-binding proteins were defined as those exhibiting at least a two-fold increase in quantitative value (QV) in HA-KERP2 or HA-KERP2 ∆185–239 compared to HA-mock and appearing in at least two out of four biological replicates. In HA-KERP2 nuclear fraction, 75 proteins were identified as double hits and 4 as triple hits, while its cytosolic fraction contained 14 double-hit and 5 triple-hit proteins (Fig. 4 A-B, Table S3). GO enrichment analysis indicated that nuclear KERP2 interactors were linked to vesicles and non-membrane-bound organelles, including ribosomes, with functional associations to binding, metabolism, and ribosome biogenesis (Fig. S3E-F). Meanwhile, KEGG enrichment analysis of cytosolic interactors highlighted their involvement in ribosome and protein processing in the endoplasmic reticulum (ER) (Fig. 4 D). To further characterize the nuclear interactome, proteins were categorized based on functionality beyond KEGG enrichment. They were grouped into clusters related to protein transport, protein folding, nucleic acid binding, and ribosome biogenesis, as visualized in a heat map of their QVs (Fig. 4 E, Table 1 ). Notably, EhRab11B, EhVPS45A, and clathrin coat assembly protein—key players in endosomal trafficking—were identified. Proteins associated with nucleic acid binding and ribosome biogenesis were more enriched in HA-KERP2, whereas those involved in protein folding and certain aspects of protein transport were more prominent in HA-KERP2 ∆185–239 . The cytosolic interactome also revealed interactions with Granin1, Granin2, and Bip, consistent with nuclear interactors, along with EhC2B (Fig. 4 F, Table 2 ). Table 1 Enriched and significant nuclear hits in HA-KERP2 Description Functionality Accession number Sjogren's syndrome/scleroderma autoantigen 1 (Autoantigen p27) EHI_006840 EhRab11b Protein Transport EHI_107250 Granin 1 Protein Transport EHI_167300 Nucleolar GTP-binding protein 1, putative Ribosome Biogenesis EHI_174940 Calmodulin, putative EHI_000130 Signal recognition particle protein SRP54, putative Protein Transport EHI_004750 Nuclear transport factor 2 domain containing protein Protein Transport/Ribosome Biogenesis EHI_035490 Transcription factor/nuclear export subunit protein 2 Nucleic Acid Binding EHI_052850 Cell division protein kinase, putative EHI_105300 Enhancer binding protein-1 Nucleic Acid Binding EHI_121780 HMG box protein Nucleic Acid Binding EHI_179340 RNA recognition motif domain containing protein Nucleic Acid Binding EHI_026440 Histone deacetylase, putative Nucleic Acid Binding EHI_119320 Chromatin organization modifier domain containing protein Nucleic Acid Binding EHI_031370 EhVPS45A Protein Transport EHI_160900 HSP70 with ER retention signals, predicted as BiP Protein Folding/ Protein Transport EHI_199590 Heat shock protein, putative Protein Folding EHI_022620 Heat shock protein 90, putative Protein Folding EHI_102270 Chaperonin containing TCP-1 delta subunit, putative Protein Folding EHI_114120 90 kDa heat shock protein, putative Protein Folding EHI_163480 Cysteine protease-C6 Proteolysis EHI_127030 14-3-3 protein (EhP2) Protein Transport EHI_098280 Clathrin coat assembly protein, putative Protein Transport EHI_135430 Granin 2 Protein Transport EHI_167310 Eukaryotic translation initiation factor 6, putative Ribosome Biogenesis EHI_006170 Nucleolar phosphoprotein Nopp34, putative Ribosome Biogenesis EHI_068680 Ribosome biogenesis regulatory protein, putative Ribosome Biogenesis EHI_098810 Guanine nucleotide-binding protein subunit beta 2-like 1, putative EHI_110400 Armadillo/beta-catenin-like repeats EHI_068510 Ras family GTPase EHI_137700 Rho family GTPase EHI_190440 Actophorin, putative EHI_197480 Actin, putative EHI_198930 WD40 repeats EHI_103620 Rab family GTPase EHI_143650 Profilin, putative EHI_176140 Serine-rich 25 kDa antigen protein EHI_116360 Protein tyrosine kinase domain-containing protein EHI_101280 Malic enzyme, putative EHI_044970 Lysyl-tRNA synthetase EHI_047810 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, putative EHI_050940 L-myo-inositol-1-phosphate synthase EHI_070720 Alcohol dehydrogenase, putative EHI_125950 phosphoglycerate kinase, putative EHI_188180 Lipase (class 3), putative EHI_032470 V-type ATPase, A subunit, putative EHI_043010 protein disulfide isomerase, putative EHI_071590 fructose-1,6-bisphosphate aldolase, putative EHI_098570 Alcohol dehydrogenase 3, putative EHI_198760 Pyruvate phosphate dikinase EHI_009530 Enolase, putative EHI_130700 26S proteasome non-ATPase regulatory subunit, putative Proteolysis EHI_030170 Ubiquitin binding Proteolysis EHI_031950 26S protease regulatory subunit, putative Proteolysis EHI_080890 Ubiquitin, putative Proteolysis EHI_083270 40S ribosomal protein S4 Ribosome EHI_118170 60S ribosomal protein L13 Ribosome EHI_181560 60S ribosomal protein L7a, putative Ribosome EHI_029530 60S ribosomal protein L18a Ribosome EHI_035600 40S ribosomal protein S7, putative Ribosome EHI_067530 60S ribosomal protein L11 Ribosome EHI_124300 60S ribosomal protein L9 Ribosome EHI_126140 Ribosomal protein eS8, putative Ribosome EHI_009870 60S ribosomal protein L2/L8, putative Ribosome EHI_127200 Uncharacterized protein EHI_155400 Uncharacterized protein EHI_155590 Uncharacterized protein EHI_017750 Uncharacterized protein EHI_085010 Uncharacterized protein EHI_124820 Uncharacterized protein EHI_155590 Uncharacterized protein EHI_178780 Uncharacterized protein EHI_183160 Uncharacterized protein EHI_189930 Uncharacterized protein EHI_194870 Uncharacterized protein EHI_005150 Uncharacterized protein EHI_053100 Uncharacterized protein EHI_178970 Uncharacterized protein EHI_197450 Uncharacterized protein EHI_122900 Table 2 Enriched and significant cytosolic hits in HA-KERP2 Description Functionality Accession number 60S ribosomal protein L12, putative EHI_030710 Heat shock protein 70, putative Protein Folding EHI_052860 ribosomal protein S25, putative EHI_074800 Grainin 1 Protein Transport EHI_167300 Acetyl-CoA synthetase, putative EHI_178960 Glyceraldehyde-3-phosphate dehydrogenase, putative EHI_008200 Uncharacterized protein EHI_017690 40S ribosomal protein S5 EHI_044590 Ras family GTPase Vesicular Trafficking EHI_058090 EhC2B EHI_059860 Nucleosome assembly protein, putative Nucleic Acid Binding EHI_072030 40S ribosomal protein S15a, putative EHI_073600 Fructose-1,6-bisphosphate aldolase, putative EHI_098570 Actin, putative EHI_107290 Ribosomal protein L11, putative EHI_124300 40S ribosomal protein S21, putative EHI_126870 Uncharacterized protein EHI_146110 Grainin 2 Protein Transport EHI_167310 70 kDa heat shock protein, putative Protein Folding EHI_199590 Given the established role of Rab11b in extracellular cysteine protease transport in E. histolytica , we performed IFA to confirm its interaction with HA-KERP2. This revealed co-localization of HA-KERP2 with Rab11B-positive vesicles (Fig. 4 G), supporting its involvement in vesicle-associated trafficking. Translocation of KERP2 from E. histolytica to host epithelial cells Since KERP1 and KERP2 were first identified as binding to the brush border of Caco-2 cells, we investigated whether KERP2 is truly transferred from E. histolytica to host cells and contributes to host-parasite interactions. Co-culturing HA-KERP2-expressing trophozoites with Caco-2 cells for 2 hours, followed by IFA, revealed punctate HA-KERP2 signals in the cytosol and nucleus of Caco-2 cells (Fig. 5 A). Live imaging of GFP-HA-KERP2-expressing trophozoites moving over the Caco-2 monolayer confirmed GFP signals within Caco-2 cells, appearing in zone 1 and zone 2 between 30 and 45 minutes (Fig. 5 B, Video S4, S5). Higher-magnification imaging revealed similar punctate structures (Fig. 5 C). This translocation was absent in control experiments using trophozoites expressing GFP-RtcB2, a cytosolic tRNA ligase (Fig. S4A-B). To validate these observations under more physiological conditions, we used a 3D crypt model with differentiated enterocytes (Fig. 5 D). After 2 hours of interaction, HA-KERP2 signals were detected in enterocytes, whereas GFP-HA remained within trophozoites (Fig. 5 E). Immunoblot analysis further confirmed KERP2 translocation. HA-KERP2-expressing trophozoites were co-cultured with a Caco-2 monolayer for 1–3 hours, and cells were washed to separate E. histolytica from Caco-2 cells (confirmed by CS-1 and GAPDH markers, Fig. 5 F). Western blotting detected HA-KERP2 in Caco-2 cells after 1 hour, with levels increased at 2 and 3 hours. Notably, galactose treatment, which inhibits E. histolytica adhesion, significantly reduced KERP2 translocation. To explore how KERP2 is released, we examined its secretion via extracellular vesicles (EVs). Full-length HA-KERP2 was undetectable in EVs, but HA-KERP2 Δ185–239 and GFP-HA were enriched, which may be due to redundant protein disposal rather than functional secretion (Fig. S4C). KERP2 Trafficking Mechanism in E. histolytica and Caco-2 Cells To investigate KERP2 trafficking during E. histolytica interaction with epithelial cells, we performed immuno-EM to visualize the localization of HA-KERP2 (Fig. 6 A). E. histolytica expressing HA-KERP2 were co-cultured with Caco-2 for 1 hour. In E. histolytica , HA-KERP2 signals were predominantly detected near the contact side with Caco-2 cells, besides the nuclear localization. In Caco-2 cells, HA-KERP2 was primarily observed at the microvillus, within the cytosol, and in endosome-like structures, suggesting active uptake. To further assess the mechanism of KERP2 uptake, we expressed and purified recombinant His-GFP-KERP2 and His-GFP in E. coli (Fig. S5A-B). Caco-2 cells were incubated with 3 µM of either His-GFP-KERP2 or His-GFP in complete EMEM, and GFP uptake was quantified over time using flow cytometry (FACS) after trypsinization (Fig. 6 B-C). Within 1 hour, GFP signals were detected in 4.33% of Caco-2 cells exposed to His-GFP-KERP2, increasing progressively to 25.2% over 24 hours, as also reflected by a rise in median fluorescence intensity (MFI). In contrast, His-GFP alone showed minimal uptake, indicating that KERP2 facilitates selective internalization. To determine whether endocytosis plays a role in KERP2 internalization, we incubated Caco-2 cells with His-GFP-KERP2 or RITC-dextran, a well-established endocytosis marker, at 35.5°C and 4°C (Fig. 6 D). As expected, RITC-dextran uptake was significantly inhibited at the lower temperature, decreasing from 53.7% at 35.5°C to 1.92% at 4°C, consistent with the suppression of endocytic activity at reduced temperatures. Similarly, His-GFP-KERP2 internalization was markedly reduced at 4°C, with GFP signals detected in only 2.25% of cells compared to 16.5% at 35.5°C, suggesting that KERP2 uptake may occur via micropinocytosis or energy-dependent endocytosis-like mechanism. To assess the persistence of internalized KERP2, we exposed Caco-2 cells to His-GFP-KERP2 or His-GFP for 24 hours, then washed the cells thoroughly with DPBS and replenished the medium (Fig. 6 E). FACS analysis at 24 and 48 hours post-wash revealed that GFP signals persisted in 11.7% and 7.2% of Caco-2 cells, respectively, suggesting that a fraction of internalized KERP2 remains undegraded or unprocessed within host cells. Since KERP2 was not detected in extracellular vesicles (EVs) but was previously identified in membrane fractions, we investigated its potential membrane association. A lipid overlay assay using recombinant His-KERP2 (Fig. S5C-D), but not His-GFP, demonstrated binding affinity for PI(3)P, PI(4)P, PI(5)P, PI(4,5)P 2 , and PA (Fig. 6 F). Functional Impact of KERP2 on Host Protein Networks and Gene Expression To investigate the impact of KERP2 on host epithelial cells, we performed co-IP coupled with MS in three biological replicates to identify KERP2-interacting proteins in Caco-2 cells. Caco-2 cells were co-cultured with HA-KERP2- or HA-mock-expressing E. histolytica trophozoites, followed by separation, lysate collection, and co-IP analysis. Significant interactors, defined by a more than 2-fold increase in HA-KERP2 samples in at least two replicates, included 78 double-hit and 5 triple-hit proteins (Fig. 7 A). GO enrichment analysis revealed associations with cadherin binding, actin binding, and transcription coactivator binding (Fig. 7 B). Functional categorization (Fig. 7 C) highlighted triple-hit interactors PPP6C, PPP6R, and RANGAP1, along with double-hit proteins such as CUL5, UBR5, and MIOS, which are linked to signaling pathways and cell cycle regulation. Several interactors were also involved in cytoskeletal organization and adhesion, including PDLIM1, PFN1, ARPC3, CNN3, LASP1, CLDN4, ITGB1, and CTNNB1. Intracellular trafficking proteins, such as EEA1, SNX5, and DCTN1, were also identified. To assess the effects of KERP2 on host gene expression, we performed RNA-seq on Caco-2 cells co-cultured with HA-KERP2-overexpressing, psAP-KERP2gs, or wild-type E. histolytica G3 strains, alongside untreated controls. PCA and volcano plots confirmed distinct clustering among all experimental groups, reflecting condition-specific transcriptional profiles (Fig. S6A-D). To specifically examine KERP2-regulated genes, we prioritized those showing significant differential expression in HA-KERP2- vs. psAP-KERP2gs-treated cells, revealing clear separation in PCA (Fig. 7 D). Differentially expressed genes (adjusted p-value < 0.05, absolute log2 fold change ≥ 1) in HA-KERP2-treated cells included upregulated pro-inflammatory mediators (IL1B, IL36G), signaling adapters (SLA), and heme-related genes (HRG), while cytoskeletal regulators (CYTIP, MYOCD), stress-response factor (NUPR1), and solute transporters (SLC15A3) were downregulated (Fig. 7 E). Conversely, psAP-KERP2gs-treated cells exhibited increased expression of extracellular matrix components (TNC), contractility regulators (MYL7), and metabolic enzymes (CYP4B1), while cilia-associated (CFAP43), solute transport (SLC2A12), and pentose phosphate pathway (TKTL1) genes were downregulated. Effects of KERP2 on DNA synthetic cycle of the host epithelial cells To evaluate the effects of KERP2 on host cell cycle progression, EdU incorporation assays were performed in Caco-2 cells following a 2-hour interaction with or without different E. histolytica strains: HA-KERP2, HA-mock, KERP2gs, and psAP-mock in the G3 background. After co-culture, E. histolytica cells were removed through washes with 2% galactose, and Caco-2 cells were incubated with EdU in complete EMEM for 6 hours. Confocal imaging showed differences in EdU-positive nuclei among experimental groups (Fig. 8 A). Quantification of EdU-positive nuclei, visualized using Hoechst 33342, showed that HA-KERP2-, HA-mock-, and psAP-mock-exposed cells, each containing exogenous or endogenous KERP2, exhibited higher EdU-positive ratios (48 ± 4.0%, 46 ± 5.3%, and 45 ± 3.3%, respectively) compared with the wild-type Caco-2 control (35 ± 4.2%; p > 0.05). By contrast, Caco-2 cells co-cultured with KERP2 knockdown (KERP2gs) E. histolytica displayed an EdU-positive ratio of 33 ± 4.3%, which was not significantly different from the wild-type control (p = 0.9445). These findings suggest that KERP2 is important for promoting Caco-2 cell proliferation, as silencing of KERP2 in E. histolytica abrogates the effect. Effects of KERP2 on cytoskeleton of the host epithelial cells To examine the impact of KERP2 on cytoskeletal organization, we stained Caco-2 cells with anti-E-cadherin (tight junctions), phalloidin 594 (F-actin), and Hoechst 33342 (nuclei), after a 2-hour interaction (Fig. 8 C). Morphological analysis revealed that HA-KERP2-cocultured Caco-2 cells displayed an elongated shape, which was also observed in differentiated enterocytes from the 3D colon-on-chip model (Fig. S7A). Quantification of Caco-2 cell shape using cell form factor and aspect ratio measurements revealed significant changes in HA-KERP2-cocultured cells but not in KERP2gs-cocultured cells compared to controls (Fig. 8 D-E). The cell form factor, which measures cellular circularity, was 0.59 ± 0.073 in untreated control cells but was significantly reduced to 0.53 ± 0.091 in HA-KERP2-cocultured cells (p < 0.05), whereas KERP2gs-cocultured cells exhibited values similar to controls (0.57 ± 0.09). Similarly, aspect ratio measurements revealed a slight but statistically significant increase in HA-KERP2-cocultured cells (1.7 ± 0.5, p < 0.05) compared to controls (1.6 ± 0.38), while KERP2gs-cocultured cells remained unchanged (1.6 ± 0.39). To assess potential alterations in Caco-2 cell adhesion, we examined F-actin organization along the basal cell surface (Fig. 8 F). KERP2gs-cocultured Caco-2 cells exhibited the formation of stress fibers, transitioning from the wild-type resting state. In contrast, HA-KERP2-cocultured cells displayed disrupted stress fiber structures, with thinner and less continuous F-actin along the cell edges, suggesting cytoskeletal remodeling. Effects of KERP2 on motility of the host epithelial cells To further investigate the impact of KERP2 on Caco-2 cell motility, we conducted a wound healing assay using 2% FBS to minimize serum-induced proliferation. Caco-2 cells were treated for 2 hours with HA-KERP2, HA-mock, KERP2gs, or psAP-mock strains, washed with 2% galactose, and subsequently monitored for gap recovery over time (Fig. 8 G). Linear regression analysis of wound closure rates revealed that KERP2gs-treated cells exhibited significantly faster migration (Y = -0.04800*X + 1.026) compared to psAP-mock-treated cells (Y = -0.04207*X + 0.9947) (Fig. 8 H). In contrast, HA-KERP2 treatment (Y = -0.03861*X + 1.048) resulted in a slower migration rate relative to HA-mock treatment (Y = -0.03870*X + 1.024). Notably, HA-KERP2-treated, HA-mock-treated, and psAP-mock-treated cells, each theoretically expressing different levels of KERP2, migrated more slowly than untreated controls (Y = -0.04339*X + 1.026), with varying degrees of reduction. Effects of KERP2 on the tight junction integrity of the host epithelial cells To evaluate the impact of KERP2 on epithelial monolayer integrity, transepithelial electrical resistance (TEER) was measured in Caco-2 cells cultured on transwell inserts and exposed to different E. histolytica strains (Fig. 9 A-C, Fig. S7B-E). TEER measurements showed that KERP2gs-cocultured cells exhibited the largest reduction in TEER, with values decreasing by 52.0 ± 4.5% after 1 hour (from 101.7 ± 5.7% to 49.7 ± 1.2%) and by 73.0 ± 4.5% after 2 hours (to 28.7 ± 1.2%). In comparison, psAP-mock-cocultured cells exhibited 25.3 ± 4.5% TEER reduction after 1 hour (from 104.3 ± 4.5% to 79.0 ± 0.0%) and 36.3 ± 4.5% after 2 hours (to 68.0 ± 1.0%). HA-KERP2-cocultured cells exhibited a 36.0 ± 3.5% TEER reduction after 1 hour (from 98.7 ± 4.0% to 62.7 ± 0.6%) and 65.7 ± 3.5% after 2 hours (to 33.0 ± 1.0%). HA-mock-cocultured cells exhibited a 32.3 ± 3.2% TEER reduction after 1 hour (from 103.3 ± 4.2% to 71.0 ± 1.0%) and 54.0 ± 3.2% after 2 hours (to 49.3 ± 1.5%). To also assess the impact of recombinant KERP2 on epithelial monolayer integrity, relative TEER (%) was measured at 0, 3, and 24 hours post-treatment with increasing concentrations of His-GFP-KERP2 (1.5 µM, 3 µM, 6 µM) and control His-GFP (3 µM, 6 µM) (Fig. 9 D, Fig. S7F-G). TEER values were normalized to baseline before measurement. After 3 hours, His-GFP-KERP2 treatment resulted in a dose-dependent TEER reduction. The 1.5 µM treatment led to a 14.9% decrease (from 117.7 ± 7.4% to 100.1 ± 6.2%), while the 3 µM treatment caused an 18.3% reduction (from 107.5 ± 7.1% to 87.8 ± 6.6%). The most significant decrease was observed with 6 µM His-GFP-KERP2, where TEER dropped by 77.7% (from 113.9 ± 2.0% to 36.2 ± 2.3%). His-GFP controls exhibited only minor reductions, with decreases of 4.6% (3 µM) and 9.7% (6 µM). At 24 hours, TEER reductions were more pronounced across all His-GFP-KERP2 treatments. The 1.5 µM concentration led to a 43.0% decrease (from 117.7 ± 7.4% to 74.7 ± 2.8%), while the 3 µM and 6 µM concentrations resulted in reductions of 44.9% and 80.3%, respectively. Control groups showed smaller reductions, with TEER decreasing by 30.1% (3 µM His-GFP) and 35.1% (6 µM His-GFP). Discussion E. histolytica heavily relies on adhesion to epithelial cells to initiate colonization and infection, a process mediated by interactions between trophozoite plasma membrane and host brush border microvilli (Seigneur et al., 2005 ). KERP2, identified alongside KERP1, was predicted to localize in the extracellular milieu, despite lacking a transmembrane domain or GPI anchor. Its high isoelectric point (pI = 9.75) and positively charged surface suggest interactions with negatively charged molecules, potentially facilitating adhesion or binding at the host-pathogen interface. Phylogenetic analysis shows KERP2 contains a conserved SAP domain, similar to DEK protein associated with chromatin remodeling and transcription regulation (Aravind and Koonin, 2000 ; Sanden and Gullberg, 2015 ). DEK proteins, conserved among multicellular eukaryotes and mammals, contrast with their sparse presence in protozoa, where only Trypanosoma brucei (TbSAP) exhibits similar SAP domain involved in gene repression (Davies et al., 2021 ; Smith et al., 2017 ). KERP2 demonstrates functional divergence within Amoebozoa, with lineage-specific evolution indicated by homolog searches across multiple kingdoms revealing no close relatives outside Entamoeba . This uniqueness is underscored by strong purifying selection shown through dN/dS analysis, suggesting conserved function with minimal adaptive changes. Searches within Amoebozoa reveal only weakly conserved KERP2-like sequences in P. pallidum and D. purpureum , indicating that while KERP2-like sequences exist, they do not function as orthologs, emphasizing a specialized role and distinct phylogenetic status of KERP2 within Entamoeba . The localization of KERP2 in E. histolytica was confirmed using multiple approaches, including IFA, live-cell imaging, immuno-EM, and co-IP. IFA with line intensity profiling demonstrated that HA-KERP2 predominantly localizes at the nuclear periphery and within the nucleoplasm, with punctate signals observed in the cytosol. In contrast, the truncated HA-KERP2 ∆185–239 variant, lacking the coiled-coil domain, was mainly detected in the cytosol, indicating that the C-terminal region is crucial for nuclear localization. Live-cell imaging using GFP-HA-tagged constructs further supported these findings, revealing strong nuclear enrichment of full-length KERP2 but cytosolic redistribution of the truncated variant. Immuno-EM confirmed the presence of HA-KERP2 in electron-dense nuclear regions, which are typically associated with chromatin-rich areas, as observed in other eukaryotic systems where electron-dense regions correspond to heterochromatin at the nuclear periphery or near nucleoli (Ralph et al., 2005 ; Zuleger et al., 2011 ). Nuclear co-IP further corroborated the nuclear presence of KERP2 by identifying key nuclear-associated binding partners, including transcription-related proteins such as enhancer binding protein-1, HMG box protein, and histone deacetylase, as well as nuclear transport proteins such as SRP54, nuclear transport factor 2 domain-containing protein, and nucleolar GTP-binding protein 1. The nuclear localization of KERP2 suggests a potential role in transcriptional regulation, particularly in chromatin organization. Given that the E. histolytica nuclear periphery may contain nucleolus (Jhingan et al., 2009 ), KERP2 could participate in ribosome biogenesis. However, the lack of a clear nucleolar marker in E. histolytica and the transcriptional changes observed upon KERP2 knockdown, as revealed by RNA-seq, led us to focus more on its potential DNA-binding activity instead. Structurally, KERP2 shares features with DEK proteins, which associate with DNA in a structure-dependent manner rather than through sequence-specific binding (Bohm et al., 2005 ; Waldmann et al., 2003 ; Waldmann et al., 2002 ). They preferentially associate with highly expressed, ubiquitous genes without binding to specific DNA motifs (Sanden et al., 2014 ), a feature that may extend to KERP2. This hypothesis is supported by chromatin immunoprecipitation (ChIP) assays, where GFP-HA-KERP2 successfully pulled down detectable amounts of DNA, whereas GFP-HA-mock controls did not. However, no specific DNA motifs were identified, with the highest observed enrichment reaching only 3.89-fold compared to input controls (Table S6). Furthermore, all enriched regions were located within coding sequences and lacked distinct motif features. These findings suggest that, similar to DEK proteins, KERP2 may primarily associate with structured chromatin regions rather than specific DNA sequences. This chromatin association is consistent with RNA-seq results, which showed significant transcriptional changes upon KERP2 knockdown, particularly in genes associated with proteolysis regulation, sulfur amino acid metabolism, and amoebiasis. Notably, cysteine synthases (EHI_024230, EHI_160930) and methionine γ-lyase (EHI_057550) were highly upregulated, suggesting a shift in sulfur metabolism, which is critical for redox balance and oxidative stress resistence in E. histolytica during host interaction (Jeelani et al., 2017 ; Tokoro et al., 2003 ). Concomitantly, virulence-associated factors such as cysteine protease (EHI_010850) and pore-forming peptides (EHI_169350, EHI_194540, EHI_15940) were also upregulated. Notably, EHI_010850 encodes an amino acid sequence with 99.7% identity to EhCP-A7 (EHI_039610). Cysteine protease activity assays further supported this regulatory role: KERP2 knockdown significantly increased both intracellular and secreted cysteine protease activity, while KERP2 overexpression resulted in a slight reduction. These findings suggest that KERP2 may function as a chromatin organizer that modulates virulence factor expression. Cysteine proteases and amoebapores are well-established virulence factors in E. histolytica , promoting host cell degradation, tissue invasion, and immune evasion (Begum et al., 2015 ; Leippe, 2014 ; Moncada et al., 2003 ; Singh et al., 2004 ). The upregulation of these virulence factors in KERP2 knockdown cells suggests that KERP2 may act as a negative regulator of virulence gene expression. In axenic E. histolytica cultures, CP-A1, CP-A2, CP-A5, and CP-A7 account for over 90% of proteolytic activity in trophozoite extracts (Irmer et al., 2009 ). Particularly, EHI_010850 and EhCP-A7 are both significantly upregulated upon contact with human colon explants (Thibeaux et al., 2013 ), hinting that KERP2-mediated repression of virulence factors could be relevant to the repression of direct host damage. A key question is whether the nuclear-cytoplasmic trafficking of KERP2 (further discussed below) serves as a regulatory mechanism that balances virulence factor expression and metabolic adaptation. We hypothesize that during host cell contact, a portion of KERP2 is translocated to host cells, leading to a reduction of its nuclear ratio in E. histolytica . This reduction in nuclear KERP2 may relieve transcriptional repression, thereby upregulating virulence genes while concurrently enhancing sulfur metabolism to support increased redox homeostasis. This dual response–activation of cysteine proteases for extracellular matrix degradation and metabolic adaptation for stress resistance–could enable E. histolytica to fine-tune its pathogenic potential in response to environmental cues. Future research should investigate whether nuclear KERP2 levels regulate these processes in a dose-dependent manner and whether its redistribution upon host interaction acts as a dynamic switch controlling both virulence and metabolic adaptation. KERP2 exhibits a dynamic intracellular distribution in E. histolytica , with immuno-EM localization revealing its presence in the nucleus, vesicular structures, and electron-dense granule (EDG)-like compartments within the cytosol. Despite being localized in cytosolic vesicles, full-length KERP2 was not detected in the EV fraction under resting conditions. co-IP analysis identified multiple trafficking-related interactors, including EhRab11B, signal recognition particle protein SRP54, EhVPS45, and clathrin coat assembly protein, suggesting that KERP2 may be transported via recycling endosomes or endosomal sorting pathways. Recycling endosomes, regulated by Rab11B, play a key role in E. histolytica vesicular trafficking and secretion of virulence factors such as cysteine proteases (Mitra et al., 2007 ). The presence of KERP2 in Rab11B-positive vesicles, but its absence in crude EV fractions, suggests that it may follow an unconventional secretion pathway, likely mediated by recycling endosome-dependent exocytosis rather than exosome release. One possible explanation for the selective secretion of KERP2 is its preferential association with the plasma membrane, potentially due to its positively charged surface (Goldenberg and Steinberg, 2010 ; Woolfson, 2023 ). Lipid overlay assays demonstrated that recombinant His-GFP-KERP2 strongly binds to phosphoinositides, including PI3P, PI4P, and PI(4,5)P2, which are enriched in endosomal and plasma membranes (Posor et al., 2022 ). This suggests that KERP2 may be retained at lipid microdomains involved in host-parasite interactions. This hypothesis is further supported by the Immuno-EM, showing KERP2 localization at the plasma membrane, particularly at the parasite-host interface, reinforcing the role of KERP2 as a surface-associated effector rather than being freely secreted. To answer a critical question whether KERP2 can be translocated into host cells during E. histolytica -epithelial interactions, IFA, live-cell imaging, and immunoblot analysis of E. histolytica -Caco-2 co-cultures were performed. HA-KERP2 and GFP-HA-KERP2 signals were detected inside Caco-2 cells and in a 3D enterocyte crypt model, appearing as punctate cytosolic structures, confirming its uptake. Notably, inhibiting E. histolytica adhesion abolished KERP2 translocation, reinforcing the contact-dependent nature of its release from E. histolytica . Immuno-EM showed HA-KERP2 within microvilli, the cytosol, and endosome-like vesicles, reinforcing that KERP2 enters the host through an active endocytic process. This was further validated by recombinant His-GFP-KERP2 uptake assays, where purified His-GFP-KERP2 induced a fluorescent increase in Caco-2 cells, quantified by FACS. Trypsinization before FACS ensured that KERP2 was truly internalized rather than merely adhering to the cell surface. Notably, uptake was significantly reduced at 4°C, indicating that KERP2 entry is energy-dependent and mediated by endocytosis. The persistence of KERP2 within punctate cytosolic structures raises the question of whether it remains in endosomal compartments or escapes into the cytosol. The retention of recombinant His-GFP-KERP2 in Caco-2 cells after 48 hours suggests that it is not simply targeted for lysosomal degradation. Instead, its interaction with host endocytic regulators (EEA1, SNX5), as revealed by HA-KERP2 co-IP in Caco-2 cells, suggests potential crosstalk with host vesicular trafficking pathways. Additionally, HA-KERP2 co-IP in Caco-2 cells identified potential receptors for endocytosis, including claudin-4 (CLDN4) and integrin beta-1 (ITGB1), both of which have been demonstrated to be exploited by pathogens for host cell internalization. CLDN4 serves as a receptor for Clostridium perfringens enterotoxin (CPE), enabling toxin binding and tight junction disruption (Sonoda et al., 1999 ), while ITGB1 has been hijacked by Streptococcus pneumoniae and human papillomavirus (HPV) to facilitate adhesion and invasion (De Gaetano et al., 2024 ; Woodham et al., 2012 ). Given that KERP2 is internalized via an endocytic pathway, these receptors may mediate its uptake, however, further validation is required. Our observation that recombinant His-GFP-KERP2 remained undegraded in Caco-2 cells for up to 48 hours suggests that KERP2 may have specific, active functions within host epithelial cells. To explore these potential roles, we performed co-IP assays, which identified a range of host proteins interacting with KERP2. These include regulators of the cell cycle and cytoskeletal organization. Complementary RNA-seq analyses showed differential expression of genes linked to stress responses and cytoskeletal remodeling in E. histolytica -exposed Caco-2 cells. In details, cells treated with HA-KERP2 exhibited upregulation of pro-inflammatory mediators and signaling adapters, alongside downregulation of cytoskeletal regulators and stress-response genes. In contrast, psAP-KERP2gs-treated cells showed elevated expression of extracellular matrix components and contractility regulators. These data point to two major pathways, cell cycle regulation and cytoskeletal dynamics, being prominently affected by KERP2. Specifically, the interaction of KERP2 with PPP6C, the catalytic subunit of protein phosphatase 6 (PP6), may underlie changes in cell cycle progression. PP6 is known to regulate DNA damage repair and inflammatory signaling (Ohama, 2019 ), while its regulatory subunits (PPP6R) stabilize PPP6C and modulate localization and substrate specificity (Ohama et al., 2013 ). Notably, PP6 levels increase with cell density in epithelial cells, where PP6 may interact with the E-cadherin cytoplasmic tail to support cell-cell adhesion (Ohama et al., 2013 ). However, the precise triggers for PP6 upregulation and its functional outcomes remain unresolved, and PPP6C deficiency appears to have opposing effects across different cell types. In mouse embryonic fibroblasts, PPP6C depletion causes G1/S arrest (Ohama et al., 2013 ), whereas in primary keratinocytes, PPP6C loss promotes S-phase entry and proliferation (Hayashi et al., 2015 ; Yan et al., 2015 ). In parallel, the interactions of KERP2 with cytoskeletal regulators provide another line of evidence for functional perturbations in actin dynamics. PFN1 (profilin 1) and ARPC3 (a subunit of the ARP2/3 complex) both have direct, well-documented roles in actin polymerization (Goley and Welch, 2006 ; Pizarro-Cerda et al., 2017 ; Rotty et al., 2013 ; Witke, 2004 ). PFN1 recharges actin monomers by promoting the exchange of ADP for ATP, while ARPC3 mediates new actin filament nucleation and branching. Additionally, LASP1 and PDLIM1, although not direct drivers of actin polymerization, act as scaffolding proteins influencing cellular migration, focal adhesion, and overall cytoskeletal organization (Orth et al., 2015 ; Zhou et al., 2021 ). From these findings, we hypothesize that the perturbation of key molecules for signaling pathways that could affect cell cycle and cytoskeletal regulation may be primary consequences of KERP2 in Caco-2 cells. Nevertheless, it is important to note that other signaling axes, such as NF-κB, mTOR, and multiple ubiquitin-mediated pathways, also emerged from the co-IP data and are likely to integrate with or modulate the effects on cell cycle and cytoskeleton. Further experimental validation of these pathways will be necessary to elucidate the full range of KERP2 influence on host cell physiology. The observed increase in EdU-positive nuclei in KERP2-expressing E. histolytica -cocultured Caco-2 cells suggests that KERP2 modulates host cell cycle progression. While knockdown of KERP2 (KERP2gs) had no detectable effect on the G1/S transition, the presence of KERP2 led to a greater proportion of cells entering S phase. One possibility is that KERP2 inhibits PP6 activity, thereby promoting proliferation; however, the precise mechanism by which PP6 regulates the cell cycle remains unclear. Alternatively, enhanced proliferation could be a downstream effect of altered signaling or disrupted cytoskeletal architecture. Consistent with these notions, RNA-seq data from HA-KERP2-treated cells showed upregulation of inflammatory mediators, signaling adapters, and downregulation of cytoskeletal regulators, stress-response genes, and solute transporters, as discussed above. Such transcriptional changes may create an environment more conducive to S-phase entry. For instance, diminished expression of CYTIP and MYOCD could relax the cytoskeletal constraints that normally govern cell cycle checkpoints (Ingber, 1993 ; Mammoto and Ingber, 2009 ), while elevated inflammatory signals (IL1B, IL36G) might activate proliferative or survival pathways (Karin and Greten, 2005 ). Nevertheless, these findings imply that KERP2 provides E. histolytica with the capacity to promote the G1/S transition in host epithelial cells, and this effect is significantly reduced when KERP2 is knocked down. Functional assays demonstrate that KERP2 disrupts host cytoskeletal dynamics. Specifically, Caco-2 cells co-cultured with HA-KERP2 exhibit an elongated shape and disrupted stress fibers, suggesting that KERP2 directly manipulates actin networks and junctional complexes. Moreover, recombinant His-GFP-KERP2 alone was sufficient to reduce TEER, indicating that cytoskeletal disruption by KERP2 does not necessarily require high levels of cysteine protease activity. In contrast, KERP2gs strain likely trigger a protease-driven route to epithelial damage. These parasites secrete elevated levels of cysteine proteases as discussed above, which degrade tight junction proteins and extracellular matrix components, leading to a pronounced TEER reduction. However, the absence of KERP2 also appears to allow host cells to mount a compensatory cytoskeletal response, as evidenced by robust stress fiber formation and faster wound closure. These findings suggest that KERP2gs-induced damage elicits a protective or reparative actin remodeling response. Thus, we hypothesized that E. histolytica employs two distinct strategies to compromise epithelial integrity: (i) direct actin disruption by KERP2 and (ii) protease-mediated tight junction. While HA-KERP2 strain secretes less proteases, they still compromise barrier function through cytoskeletal alterations. Conversely, KERP2 -silenced parasites cause significant junctional damage via proteases but inadvertently trigger stronger host actin-mediated repair mechanisms. These observations highlight KERP2 as a key effector that modulates the mode of epithelial barrier disruption. Future studies should investigate the regulatory circuits controlling KERP2 expression and protease secretion and assess whether cytoskeletal remodeling enhances parasite invasiveness or reflects an adaptive host countermeasure. Overall, our study identifies KERP2 as a dual-function effector that regulates both E. histolytica homeostasis and host epithelial responses, shedding light on its multifaceted role in parasitism and virulence. By simultaneously disrupting actin networks and junctional complexes while modulating cysteine proteases levels, KERP2 fine-tunes a balance between cytoskeletal perturbation and protease-mediated degradation to evade host defenses. These findings reveal how E. histolytica employs both direct cytoskeletal manipulation and protease-driven mechanisms to compromise epithelial barrier integrity and establish infection. Beyond amoebiasis, this study provides a broader framework for understanding how extracellular pathogens utilize multifunctional effectors to regulate virulence and manipulate host responses. The discovery of KERP2’s dual roles highlight potential parallels in other pathogens that fine-tune their pathogenicity by integrating host manipulation with self-transcriptional control. Future research into the regulatory mechanisms governing KERP2 function could yield valuable insights into host-pathogen interactions and inform the development of targeted therapeutic strategies. Methods Structural Modeling, Domain Identification, and Biochemical Property Analysis The three-dimensional structure of KERP2 was modeled using I-TASSER (Yang and Zhang, 2015 ; Zheng et al., 2021 ; Zhou et al., 2022 ). Structural visualization and refinement were performed using PyMOL (The PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC.). Secondary structural motifs, including a coiled-coil domain spanning amino acids 178–216, were predicted using Marcoil and PCOILS (Delorenzi and Speed, 2002 ; Gabler et al., 2020 ; Lupas, 1996 ; Lupas et al., 1991 ). The SAP domain of KERP2 was manually aligned based on residue conservation patterns described in (Aravind and Koonin, 2000 ). Automated alignment attempts using tools such as CLC Viewer (QIAGEN Bioinformatics, Aarhus, Denmark) and PSI-BLAST did not yield satisfactory results due to the weak sequence conservation of the SAP domain. To address this, residues in KERP2 were manually examined and aligned to known SAP domains following the consensus criteria outlined in the reference study. Key features used for the alignment included: hydrophobic or aliphatic residues (YFWLIVMA), small residues (SAGTVPNHD), polar residues (STQNEDRKH), and bulky residues (KREQWFYLMI). The alignment process was further varified by predicted secondary structure features by PHD program (Rost and Sander, 1994 ). Nuclear localization signals (NLS) were mapped using the NLS Mapper tool (Kosugi et al., 2009 ). The isoelectric point (pI) of KERP2 was calculated using ExPASy’s Compute pI/Mw tool (Gasteiger et al., 2003 ). Surface charge distribution was analyzed by combining the predicted structure from I-TASSER with visualization and electrostatics tools in VMD and APBS (Humphrey et al., 1996 ; Jurrus et al., 2018 ). Phylogenetic Analysis BLASTp and HMMER searches were performed to identify KERP2 orthologs across a broad range of taxa using KERP2 as a query. The obtained sequences were aligned using MAFFT v7.475 (Katoh and Standley, 2013 ) and trimmed by TrimAl v1.4 (Capella-Gutierrez et al., 2009 ). Phylogenetic reconstruction was conducted using the maximum likelihood method in IQ-TREE v2.3.6 (Minh et al., 2020 ) with default parameters. The resulting phylogenetic tree was visualized using iTOL v7.0 (Letunic and Bork, 2024 ). The ratio of nonsynonymous (dN) to synonymous (dS) substitutions (ω = dN/dS) was calculated using the codeml module in PAML v4.9. Plasmid Construction Total RNA from E. histolytica trophozoites was extracted using the TRIzol reagent (Invitrogen) following the manufacturer’s protocol. Briefly, cells were lysed directly in TRIzol reagent, and the homogenized lysate was incubated at room temperature for 5 minutes to allow complete dissociation of nucleoprotein complexes. Chloroform (0.2 mL per 1 mL of TRIzol) was added to the lysate, vortexed vigorously for 15 seconds, and incubated at room temperature for 3 minutes. The mixture was centrifuged at 12,000 × g for 15 minutes at 4°C to separate phases. The aqueous phase containing RNA was transferred to a new tube, and RNA was precipitated by adding isopropanol (0.5 mL per 1 mL of TRIzol), incubated at room temperature for 10 minutes, and centrifuged at 12,000 × g for 10 minutes at 4°C. The RNA pellet was washed with 75% ethanol (1 mL per 1 mL of TRIzol) and centrifuged at 7,500 × g for 5 minutes at 4°C. After air-drying, the pellet was dissolved in RNase-free water and quantified using a NanoDrop spectrophotometer (ThermoFisher). Messenger RNA (mRNA) was purified, and cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System (Invitrogen, ThermoFisher) according to the manufacturer’s protocol. The protein-coding region of KERP2 was amplified by PCR from E. histolytica cDNA using specific oligonucleotides summarized in Table S1 . To express KERP2 fused with an HA tag at the amino terminus in E. histolytica trophozoites, PCR fragments were digested with XmaI and XhoI, purified, and ligated into XmaI- and XhoI-digested pEhExHA vector (Nakada-Tsukui et al., 2009 ) using cohesive ends, producing the plasmid pEhEx-HA-KERP2. To generate deletion and fusion constructs: pEhEx-HA-KERP2 ∆185–239 : Reverse PCR was performed on pEhEx-HA-KERP2 using paired primers, followed by blunt-end ligation with T4 Polynucleotide Kinase and T4 DNA Ligase. pEhEx-GFP-HA-KERP2: GFP was amplified from the pEhEx-GFP vector (Nakada-Tsukui et al., 2009 ) with BglII end, then ligated into pEhEx-HA-KERP2 digested with BglII. pEhEx-GFP-HA-KERP2 ∆185–239 : GFP was amplified from pEhEx-GFP and inserted into pEhEx-HA-KERP2 ∆185–239 same as described above. To silence KERP2 in E. histolytica using antisense small RNA, a 420 base-pair fragment corresponding to the 5’ sequence of KERP2 was amplified by PCR from cDNA using appropriate primers. The amplified fragment was digested with StuI and SacI and cloned into StuI/SacI-digested psAP2-Gunma vector (Mi-Ichi et al., 2011), generating the plasmid psAP2-KERP2gs. To express recombinant KERP2, GFP-KERP2 and GFP with a histidine tag at the amino terminus in bacteria, the KERP2 coding region was first inserted into pEhEx-GFP using appropriate primers with XmaI and XhoI, following the same procedure as described for the construction of pEhEx-HA-KERP2. PCR was then performed on the resulting pEhEx-GFP-KERP2 construct to amplify the GFP-KERP2 fragment, which was subsequently inserted into the pET-151 vector using the In-Fusion HD Cloning Kit (Clontech Laboratories, CA, USA), generating pET-151-GFP-KERP2. A 5× GA linker with XmaI ends, synthesized by FASMAC Co., Ltd. (Kanagawa, Japan), was inserted between GFP and KERP2. Finally, reverse PCR on pET-151-GFP-KERP2 using specific primers was performed to construct pET-151-GFP. All constructs were validated by Sanger sequencing to ensure accurate cloning. Cell Culture and Transfection Trophozoites of the E. histolytica strain HM-1:IMSS cl-6 (Diamond et al., 1978 ) and G3 (Bracha et al., 2006 ) were maintained axenically in Diamond’s BI-S-33 medium (Diamond et al., 1978 ) at 35.5°C. Plasmids encoding HA-tagged and GFP-HA-fused KERP2 or its variants, generated as described above, were introduced into HM-1 or G3 trophozoites via lipofection, following the protocol established by (Nozaki et al., 1999 ). Transfected trophozoites were selected and maintained in medium containing 10 µg/mL of G418 (#11811031, Gibco/Life Technologies, Waltham, MA, USA). Plasmids for gene silencing experiments were introduced into the G3 strain by the same lipofection method, and the transfectants were also maintained in medium supplemented with 10 µg/mL of G418. Protein expression was verified by western blotting as described below. Caco-2 cells (ATCC) were cultured in Eagle's Minimum Essential Medium (EMEM; ATCC 30-2003) supplemented with 20% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA.) as complete EMEM. The medium was replaced every two days, and cells were incubated under standard cell culture conditions at 37°C in a humidified atmosphere with 5% CO₂. Establishment of 3D Crypt Model The 3D crypt model was established by following (Hinman et al., 2021 ). Primary intestinal crypts were first isolated and cultivated in collagen-based scaffolds. Crypts were initially dissociated from intestinal tissue using a crypt isolation buffer containing EDTA and dithiothreitol (DTT). The suspension was centrifuged at 600 g for 1 minute, and the pellet was resuspended in pre-warmed maintenance medium (MM) at a concentration of 240 crypts/mL. Crypts were seeded into 6-well plates pre-coated with neutralized collagen hydrogels, which were prepared by combining acid-solubilized collagen with a neutralization buffer (pH 7.4) and allowing the hydrogels to set for 1 hour at 37°C in a humidified CO₂ incubator. The seeded crypts were cultured in MM at 37°C with medium changes every 48 hours until confluency (~ 80%) was achieved. Upon confluency, the cells were passaged by mechanically fragmenting the crypt clusters using a double pipette tip technique and reseeded into fresh collagen-coated scaffolds. To construct the 3D crypt arrays, micromolded collagen scaffolds were fabricated using PDMS molds to mimic the architectural features of in vivo intestinal crypts. These scaffolds were crosslinked and coated following established protocols to ensure mechanical stability and optimal cell attachment. Crypts were seeded into these scaffolds in differentiation medium supplemented with 10 µM Y-27632 to enhance cell survival and attachment. Cultures were maintained in a humidified CO₂ incubator at 37°C, with media changes every 48 hours. Differentiation was induced by establishing a growth-factor gradient across the 3D scaffolds. WRN medium was supplied to the basal reservoir, and differentiation medium was added to the luminal reservoir to promote compartmentalization of stem/proliferative and differentiated cells. The gradient was maintained for 4 days, enabling the formation of a spatially organized crypt-villus axis. After differentiation, the 3D crypts were prepared for imaging and functional analyses. The scaffolds were compatible with confocal microscopy and other phenotypic assays, ensuring their utility for downstream applications. Immunofluorescence Assays (IFA) Immunofluorescence assays were performed to analyze the localization of KERP2 and its variants in E. histolytica or mammalian cells. E. histolytica cells were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 minutes at room temperature, followed by permeabilization and blocking with 0.2% saponin and 1% bovine serum albumin (BSA) in PBS for 10 minutes. Cells were incubated with primary antibodies, including anti-HA (1:200 dilution, clone 11MO; Covance, Princeton, NJ, USA.), anti-CS1 (1:500 dilution), or anti-Rab11b (1:500 dilution), for 1 hour at 4°C. After PBS washes, samples were stained with Alexa Fluor™ 488 Goat anti-Mouse IgG (H + L) and Alexa Fluor™ 594 Goat anti-Rabbit IgG (H + L) (Thermo Fisher Scientific, Waltham, MA, USA) at 1:1000 dilution, along with Hoechst 33342 (1:8000 dilution; Thermo Fisher Scientific) for 1 hour at room temperature in the dark. Caco-2 monolayers, cultured for 21 days on Millicell EZ slides (Merck Millipore, Burlington, MA, USA), were co-cultured with E. histolytica HA-KERP2 or GFP-HA-KERP2-expressing strains for 2 hours. Following interaction, cells were fixed with 4% PFA in PBS for 20 minutes, then permeabilized and blocked with 0.2% saponin, 1% BSA, and 50 mM glycine in PBS for 2 hours. Samples were incubated overnight at 4°C with anti-HA (1:200 dilution, clone 11MO; Covance). After washing, cells were stained with Alexa Fluor™ 488 Goat anti-Mouse IgG (H + L) (Thermo Fisher Scientific) at 1:1000 dilution, with or without Alexa Fluor™ 594 Phalloidin (1:400 dilution; Thermo Fisher Scientific), and Hoechst 33342 (1:8000 dilution; Thermo Fisher Scientific) for 1 hour at room temperature in the dark. E. histolytica GFP-HA-KERP2 strains were pre-stained with CellTracker Deep Red (Thermo Fisher Scientific) in Opti-MEM (Thermo Fisher Scientific) for 1 hour. Parasites were seeded onto the 3D-crypt enterocyte model and allowed to interact for 2 hours. Immunofluorescence staining followed the same protocol as for Caco-2 cells. For Caco-2 morphology observation after treatment with E. histolytica transformants, the same IFA process was used. Primary antibody anti-E-cadherin (1:1000 dilution, clone 3195; Cell Signaling Technology, Danvers, MA, USA) and secondary antibody Alexa Fluor™ 488 Goat anti-Rabbit IgG (H + L) (Thermo Fisher Scientific) were used together with Alexa Fluor™ 594 Phalloidin (1:400 dilution; Thermo Fisher Scientific) and Hoechst 33342 (1:8000 dilution; Thermo Fisher Scientific). Imaging was performed using an FV3000 confocal microscope (EVIDENT) for E. histolytica and Caco-2 cells, and a Dragonfly High-Speed Confocal Microscope System (Oxford Instruments, Abingdon, Oxfordshire, UK) for 3D-crypt enterocytes. Image processing and line intensity profile analyses were conducted using Imaris 10.2 and Fiji software (Schindelin et al., 2012 ). Live Imaging Live-cell imaging was conducted to observe the localization and dynamics of GFP-HA-tagged KERP2 constructs in real time. For imaging, cells were pre-stained with Hoechst 33342 (Thermo Fisher Scientific) in Opti-MEM (Thermo Fisher Scientific) for 40 minutes and maintained in complete BI-S33 medium at 35.5°C. Live cells were plated on glass-bottom dishes and allowed to settle for 30 minutes before imaging. Imaging was performed using an LSM 780 confocal microscope (Carl Zeiss Microscopy, White Plains, NY, USA) equipped with a temperature-controlled chamber set to 35.5°C and a 63× oil-immersion objective. Time-lapse images were captured continuously for up to 5 minutes. For KERP2 transfer studies, 21-day cultured Caco-2 monolayers established on glass-bottom dishes were pre-stained with Hoechst 33342 (Thermo Fisher Scientific) in EMEM (ATCC) for 40 minutes. GFP-HA-KERP2-expressing E. histolytica was added to the Caco-2 monolayers and incubated for 30 minutes. Live imaging was performed as described above, with time-lapse images captured continuously for up to 30 minutes. Z-stack scanning was performed following time-lapse imaging. Image acquisition and analysis were conducted using Imaris 10.2 (Oxford Instruments) and Fiji software. Immuno-electron Microscopy Trophozoites overexpressing HA-KERP2 and HA-mock were cultured in BIS medium and incubated with gold disks at 35.5°C overnight to facilitate attachment. The disks carrying attached amoebae were rapidly frozen in liquid propane at − 175°C. Freeze substitution was carried out in 2% tannic acid in ethanol with 2% distilled water at − 80°C for 48 hours. The samples were then gradually warmed to − 20°C for 4 hours and subsequently to 4°C for 1 hour. Dehydration was performed with three changes of anhydrous ethanol at 4°C for 30 minutes each. Dehydrated samples were infiltrated with a 50:50 mixture of ethanol and resin (LR White; London Resin Co. Ltd., Berkshire, UK) at 4°C for 30 minutes, followed by three changes of 100% LR White resin at 4°C for 30 minutes each. Samples were transferred to fresh 100% resin and polymerized at 50°C overnight. Polymerized samples were sectioned into ultra-thin slices (70 nm) using a diamond knife on an ultramicrotome (Ultracut UCT; Leica Microsystems, Vienna, Austria). Sections were mounted on nickel grids and incubated overnight at 4°C with a primary antibody (anti-HA) diluted in 1% BSA/PBS. Grids were washed three times with 1% BSA/PBS for 1 minute each, then incubated for 2 hours at room temperature with a secondary antibody conjugated to 15 nm gold particles (goat anti-mouse IgG pAb). After secondary antibody incubation, grids were washed with PBS and fixed with 2% glutaraldehyde in 0.1 M phosphate buffer. Finally, grids were dried and stained with 2% uranyl acetate for 10 minutes, followed by lead stain solution (Sigma-Aldrich Co., Tokyo, Japan) at room temperature for 3 minutes. Samples were observed using a transmission electron microscope (JEM-1400Plus; JEOL Ltd., Tokyo, Japan) operating at an acceleration voltage of 100 kV. Digital images (3296 × 2472 pixels) were captured using a CCD camera (EM-14830RUBY2; JEOL Ltd., Tokyo, Japan). qRT-PCR and RNA-seq Total RNA was extracted from KERP2gs or psAP-mock trophozoites, and complementary DNA (cDNA) was synthesized as described in previous methods. The efficiency of KERP2 silencing in trophozoites was validated using quantitative real-time PCR (qRT-PCR). RNA polymerase II (EHI_056690) was used as the internal control. Primers were designed to amplify a 493 bp segment of KERP2 and a 204 bp segment of RNA polymerase II. Twenty-fold diluted cDNA from each strain was used as a template in reactions performed with the Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), following the manufacturer’s protocol. qRT-PCR was conducted on a StepOne Plus Real-Time PCR System (Applied Biosystems), and data analysis was performed using the DataAssist software (Thermo Fisher Scientific). RNA-seq was performed an Illumina NovaSeq 6000 platform (Fujifilm, Japan) similarly as described below. For co-culture experiments, total RNA was extracted from Caco-2 cells interacting with HA-KERP2-expressing E. histolytica G3 strain, psAP-KERP2gs trophozoites, or G3 control trophozoites after 4 hours of co-culture in complete EMEM medium. RNA integrity was assessed using an Agilent 2100 Bioanalyzer, and sequencing libraries were prepared using a strand-specific RNA-seq library preparation kit. Libraries were sequenced on an Illumina NovaSeq X Plus (Novogene, China), generating paired end reads. Sequence reads were quality-checked using FastQC (v0.11.8) and trimmed using Trimmomatic (v0.39) (Bolger et al., 2014 ). Trimmed reads were aligned to the E. histolytica reference genome assembly (AmoebaDB-68) or the human reference genome (GRCh38.p14) using STAR (v2.7.10a) (Dobin et al., 2013 ) with default parameters and gene annotation provided in the corresponding GTF file. Mapped reads were indexed and sorted using Samtools (v1.15.1) (Danecek et al., 2021 ). Read count quantification at the gene level was performed using FeatureCounts (v2.0.1) (Liao et al., 2014 ) with strand-specific parameters. Differential gene expression analysis was conducted using the DESeq2 R package (v1.36.0) (Love et al., 2014 ), with three biological replicates per condition. Gene expression normalization was performed using DESeq2’s median-of-ratios method. Log2 fold changes and adjusted P-values were computed using the Benjamini-Hochberg method to control the false discovery rate (FDR). Genes with an adjusted P-value (padj) ≤ 0.05 and an absolute log2 fold change ≥ 1 were classified as differentially expressed. Principal component analysis (PCA) and hierarchical clustering were performed to assess sample variance. Volcano plots and heatmaps were generated using ggplot2 (v3.5.1) (Wickham and SpringerLink (Online service), 2016), ggrepel (v0.9.5) (Slowikowski, 2024 ), and pheatmap (v1.0.12) (Kolde, 2018 ) to visualize differentially expressed genes across conditions. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted using clusterProfiler (v4.2.2) (Stirling et al., 2021 ) or ShinyGO (v0.82) (Ge et al., 2020 ) to identify functional categories associated with differentially expressed genes. Growth Kinetics Approximately 3 × 10⁴ exponentially growing trophozoites of the E. histolytica G3 strain, transformed with either psAP-KERP2gs or psAP-mock (control), were inoculated into 6 mL of fresh BI-S-33 medium supplemented with 10 µg/mL G418. The cultures were incubated under standard conditions, and trophozoites were counted every 24 hours using a hemocytometer to assess growth over time. Cysteine Protease (CP) Activity Assay Approximately 1.2 × 10⁶ E. histolytica transformants were cultured in 500 µL of Transfection Medium (Opti-MEM supplemented with 5 mg/mL L-cysteine and 1 mg/mL ascorbic acid) at 35.5°C for 1 hour. After incubation, cells were aliquoted into 50 µL samples and centrifuged at 3000 × g for 3 minutes. The supernatant was subsequently centrifuged at 13,000 × g for 5 minutes to collect the released form of cysteine protease (CP) activity. The cell pellet was lysed in 100 µL of PBS using three freeze-thaw cycles, and cellular debris was removed by centrifugation at 15,000 × g for 5 minutes. The resulting supernatant from this step was collected as the intracellular form of CP activity. Both released and intracellular samples were pre-incubated in assay buffer (0.1 M KH₂PO₄, pH 6.1, 1 mM EDTA, 2 mM DTT) at room temperature for 15 minutes to activate pro-forms of CP. Following activation, 75 µL of the enzyme mixture was combined with Benzyloxycarbonyl-L-arginyl-L-arginine 4-methylcoumaryl-7-amide (#3123-v, Peptide Institute, Osaka, Japan) to achieve a final substrate concentration of 10 mM. Fluorescence emission at 460 nm (excitation at 355 nm) was measured for 1 hour using a SpectraMax Paradigm multimode microplate reader (Molecular Devices, San Jose, CA, USA). 7-amino-4-methylcoumarin (#3099-v, Peptide Institute) was used as a standard to quantify specific CP activities. Results were expressed as fluorescence intensity corresponding to the production of 4-methylcoumaryl-7-amide per milligram of lysate protein. Statistical significance was evaluated using two-way ANOVA. Nuclear Fractionation and Co-immunoprecipitation Nuclear protein fractions were prepared from approximately 2 × 10⁷ cells of E. histolytica HA-KERP2, HA-KERP2 ∆185–239 , and HA-mock strains. Prior to nuclear fractionation, cells were cross-linked with 0.8 mg/mL Pierce dithiobis (succinimidyl propionate) (DSP) (Thermo Fisher Scientific) in PBS to stabilize protein-protein interactions. Nuclear protein extraction was conducted using the Nuclear Complex Co-IP Kit (#54001, Active Motif, Carlsbad, CA, USA) according to the manufacturer’s protocol. Extracted nuclear fractions were treated with the enzymatic cocktail reagent included in the kit, followed by nucleic acid digestion to degrade DNA and RNA, ensuring a focus on protein-protein interactions. The nuclear extracts were diluted in the IP low buffer provided in the kit, supplemented with protease and phosphatase inhibitors. Cytosolic fractions were also prepared for comparative analysis. Diluted nuclear and cytosolic fractions were incubated separately with anti-HA antibody (clone 11MO, Covance) overnight at 4°C. Antibody-bound complexes were captured by incubating the samples with 45 µL of pre-washed Dynabeads™ Protein A (Thermo Fisher Scientific) for 1 hour at 4°C. To minimize non-specific interactions, beads were washed three times with 500 µL of IP low buffer containing BSA, followed by three additional washes with 500 µL of IP low buffer without BSA. Bound proteins were eluted by incubating the beads with 0.3 mg/mL HA peptide (#I2149, Sigma-Aldrich) overnight at 4°C. Eluted samples were collected for downstream phosphorylation validation, western blot analysis, or mass spectrometry-based identification. Extracellular Vesicle Isolation Approximately 50 mL of serum-deprived culture medium from E. histolytica transformants was collected and centrifuged at 1,000 × g for 10 minutes at 4°C to remove intact cells. The resulting supernatant was filtered through a 0.22 µm membrane using a 50 mL syringe to eliminate larger debris. The filtered medium was then concentrated using a 100 kDa MWCO Amicon filter (Merck Millipore) at 4,000 × g for 15 minutes at 4°C. The concentrated cell-free medium was transferred to an ultracentrifuge tube and subjected to ultracentrifugation at 100,000 × g for 75 minutes at 4°C to pellet extracellular vesicles (EVs), including exosomes. The EV pellet was washed with 1× PBS, followed by a repeat of the ultracentrifugation step under identical conditions. After discarding the supernatant, the final EV pellet was resuspended in 50 µL of 1× PBS. For EV lysis, 1% Triton X-100 was added to the resuspended pellet, and the mixture was incubated on ice for 30 minutes. Preparation of Recombinant Proteins Plasmids pET-KERP2, pET151-GFP-KERP2, and pET151-GFP, designed to express recombinant His-GFP-KERP2 and His-GFP proteins, respectively, were introduced into E. coli BL21 (DE3) cells by heat shock at 42°C for 1 minute. Transformed E. coli cells were initially grown in 100 mL of Luria Bertani (LB) medium supplemented with 100 µg/mL carbenicillin at 37°C. The overnight culture was then used to inoculate 500 mL of fresh LB medium, and the cells were grown at 37°C with shaking at 200 rpm. When the optical density at 600 nm (A600) reached 0.4, the cultures were chilled on ice, and 2% ethanol and 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) were added to induce protein expression. Cultivation was continued for an additional 16 hours at 18°C. Induced E. coli cells were harvested by centrifugation at 8,000 × g for 10 minutes at 4°C. The cell pellets were washed with Tris-saline buffer (10 mM Tris-HCl, pH 7.6, and 150 mM NaCl) and resuspended in 40 mL of lysis buffer containing 50 mM Tris-HCl (pH 7.6), 250 mM NaCl, 10% (w/v) sucrose, 100 µg/mL lysozyme, 0.5 µg/mL E64, 1× cOmplete Mini Protease Inhibitor Cocktail (Roche), and 1 mM phenylmethylsulfonyl fluoride (PMSF). The suspension was incubated at room temperature for 20 minutes and lysed using a French press. Triton X-100 was added to a final concentration of 0.1% (v/v), and the lysate was centrifuged at 25,000 × g for 30 minutes at 4°C to remove cell debris. The clarified supernatant was incubated with 1 mL of 50% Ni²⁺-NTA His-Bind resin slurry at 4°C for 1 hour with gentle rotation. The resin was transferred to a column and washed three times with wash buffer containing 50 mM Tris-HCl (pH 7.6) and 2 M KCl. Bound proteins were eluted using elution buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 10% (v/v) glycerol, and 10–750 mM imidazole. The integrity and purity of the recombinant proteins were confirmed by 12% SDS-PAGE, followed by Coomassie Brilliant Blue staining or western blot analysis. Protein–lipid overlay assays The soluble fraction of recombinant His-KERP2 or His-GFP was obtained as described above and used to probe a P-6001 phospholipid membrane strip (Echelon Biosciences, Salt Lake City, UT, USA). The membrane was blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 2 hours at room temperature (RT) before incubation with 1 µg/mL of recombinant protein in PBS containing 3% BSA for 16 hours at 4°C. Following incubation, the membrane was washed three times with PBS containing 0.1% Tween-20 (PBS-T) and then incubated with an anti-His primary antibody (His-Tag (27E8) Mouse mAb #2366, Cell Signaling Technology, 1:1000) in PBS with 3% BSA for 2 hours at RT. After additional washes, the membrane was incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (Cat# 32430, Invitrogen, Thermo Fisher Scientific, 1:5000) in PBS with 3% BSA for 1 hour at RT. The blot was washed, developed using a chemiluminescent HRP substrate (Merck Millipore), and visualized using a ChemiDoc™ Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). Sample Preparation for KERP2 Translocation in Caco-2 Cells Confluent Caco-2 cells cultured in T75 flasks (~ 5 × 10⁶ cells per flask) were co-cultured with E. histolytica HA-KERP2 strains at a 5:1 ratio (Caco-2: E. histolytica ) for 1, 2, and 3 hours in complete EMEM (ATCC). For control experiments, E. histolytica HA-mock strains were co-cultured with Caco-2 cells for 1 hour. An additional experimental group involved pretreatment of E. histolytica HA-KERP2 strains with 2% galactose in complete Diamond’s BI-S-33 medium for 30 minutes prior to co-culture. During the interaction for this group, 2% galactose was also added to the complete EMEM for a 1-hour incubation. After co-culture, the mixtures were washed three times with pre-cooled 2% galactose in PBS (Thermo Fisher Scientific) to remove E. histolytica cells. The remaining Caco-2 cells were collected, while E. histolytica cells were processed separately. Both cell types were lysed using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific), supplemented with cOmplete™ Protease Inhibitor Cocktail (Roche). The lysates were subjected to western blotting for the detection of KERP2 translocation. Anti-HA antibody (ab9110, Abcam, Cambridge, UK) was used to enhance detection sensitivity during western blot analysis. Caco2 Co-immunoprecipitation Confluent Caco-2 cells grown in T75 flasks (~ 5 × 10⁶ cells per flask) were used to interact with E. histolytica HA-KERP2 or HA-mock strains at a 5:1 ratio (Caco-2: E. histolytica ) for 1 hour. Following interaction, six flasks of co-cultures were washed three times with pre-cooled 2% galactose in PBS to remove E. histolytica cells. The remaining Caco-2 cells were harvested using a cell scraper. The collected Caco-2 cells were cross-linked with 0.8 mg/mL Pierce DSP (ThermoFisher Scientific) in PBS for stabilization of protein-protein interactions. After cross-linking, cells were lysed using M-PER Mammalian Protein Extraction Reagent (ThermoFisher Scientific) supplemented with a cOmplete protease inhibitor cocktail (Roche). Lysates were incubated with anti-HA antibody (clone 11MO; Covance) overnight at 4°C. The antibody-bound complexes were captured by incubation with 40 µL of pre-washed Dynabeads Protein A (Invitrogen, ThermoFisher) for 1 hour at 4°C. Beads were washed three times with Washing Buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, 0.1% BSA) and three additional times with Washing Buffer lacking BSA to minimize non-specific interactions. Bound proteins were eluted by incubating the beads with 0.3 mg/mL HA peptide (Sigma-Aldrich) overnight at 4°C. Eluted samples were collected and prepared for downstream analyses. Flamingo Gel Staining Co-IP samples were separated by SDS-PAGE as described in the relevant methods sections. Following electrophoresis, gels were subjected to Flamingo fluorescent gel staining (Bio-Rad Laboratories) to visualize protein elution in nuclear co-IP and Caco-2 co-IP pull-down samples. The staining was performed according to the manufacturer’s protocol. Stained gels were imaged using a ChemiDoc imaging system (Bio-Rad Laboratories) to detect protein bands. Western Blotting Protein samples were separated by SDS-PAGE or Phos-tag™ SDS-PAGE (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and transferred onto PVDF membranes (Merck Millipore) using a semi-dry transfer system. Membranes were blocked with 5% non-fat milk in TBST (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) for 30 minutes at room temperature. Blocked membranes were incubated overnight at 4°C with primary antibodies, including anti-HA (1:1000, clone 11MO, BioLegend, San Diego, CA, USA), anti-CS1 (1:1000, in-house source), anti-GAPDH (1:5000, AM4300, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), anti-Histone H3 (1:1000, ab1791, Abcam), or anti-His (1:1000, #2366, Cell Signaling Technology, Danvers, MA, USA). After primary antibody incubation, membranes were washed three times with TBST and then incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Secondary antibodies used included Donkey anti-Rabbit IgG (H + L) Cross-Adsorbed, HRP (31458, Invitrogen, Thermo Fisher Scientific) and Goat anti-Mouse IgG (H + L), HRP (32430, Invitrogen, Thermo Fisher Scientific). Following secondary antibody incubation, membranes were washed three more times with TBST. Signals were detected using a chemiluminescence HRP substrate (Merck Millipore) and visualized using a ChemiDoc™ Imaging System (Bio-Rad Laboratories). Mass Spectrometry Protein extracts were buffer-exchanged using SP3 paramagnetic beads (Cytiva, Marlborough, MA, USA). Samples were rehydrated in 100 µL of 10 mM triethylammonium bicarbonate (TEAB) containing 1% SDS. Disulfide bonds were reduced with 10 µL of 50 mM dithiothreitol (DTT) at 60°C for 1 hour. After cooling to room temperature (RT), the pH was adjusted to approximately 7.5, and alkylation was performed by adding 10 µL of 100 mM iodoacetamide (Sigma-Aldrich) and incubating in the dark at RT for 15 minutes. SP3 beads (100 µg; 2 µL of 50 µg/µL) were added to the samples, followed by the addition of 120 µL of 100% ethanol. Samples were incubated at RT with shaking for 5 minutes to bind proteins to the beads. Beads were then washed three times with 180 µL of 80% ethanol. On-bead protein digestion was performed overnight at 37°C using trypsin (Pierce Trypsin, Thermo Fisher Scientific, Waltham, MA, USA; 1 µg enzyme per sample). After digestion, the supernatant was removed, dried, and rehydrated in 2% acetonitrile/0.1% formic acid for mass spectrometry analysis. Peptide analysis was performed by reverse-phase chromatography-tandem mass spectrometry (LC-MS/MS) using an EasyLC 1100 UPLC system interfaced with an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a 75 µm × 15 cm column (PicoFrit Self-pack emitter, New Objective, Woburn, MA, USA) packed in-house with ReproSIL-Pur-120-C18-AQ resin (3 µm, 120 Å bulk phase, Dr. Maisch GmbH, Ammerbuch, Germany). Separation was achieved using a 0–100% acetonitrile gradient in 0.1% formic acid over 90 minutes at a flow rate of 300 nL/minute. Survey scans of precursor ions were acquired over a range of 350–1800 m/z with a resolution of 120,000 at 200 m/z, an automatic gain control (AGC) target of 4 × 10⁵, and an RF lens setting of 45%. Internal mass calibration was used for accuracy. Precursor ions were individually isolated within a 1.5 m/z window using data-dependent acquisition with a 15-second dynamic exclusion. Fragmentation was performed using higher-energy collision dissociation (HCD) at a collision energy of 30. Fragment ions were analyzed at a resolution of 30,000 with an AGC target of 1 × 10⁵. The raw data files were analyzed using Mascot software (Matrix Science, London, UK; version 2.8.2) with the UniProt E. histolytica database (taxon ID: 294381; 20,546 entries). Searches assumed trypsin as the digestion enzyme, with a fragment ion mass tolerance of 10.0 ppm and a parent ion mass tolerance of 5.0 ppm. Carbamidomethylation of cysteine was specified as a fixed modification, while deamidation of asparagine and glutamine, oxidation of methionine, phosphorylation of serine, threonine, and lysine, and formylation of lysine and the protein N-terminus were defined as variable modifications. Peptide identifications were validated and processed using Scaffold software (Proteome Software Inc., Portland, OR, USA). PeptideProphet was used for peptide validation with a 1% false discovery rate (FDR), while ProteinProphet was used for protein inference with a confidence threshold of 95%. EdU Incorporation Assay Four-day-cultured Caco-2 cells were co-cultured with 1 × 10⁴ E. histolytica transformants at a ratio of approximately 10:1 (Caco-2: E. histolytica ) for 2 hours. After interaction, the co-cultures were washed three times with pre-cooled 2% galactose in PBS to remove E. histolytica transformants. To assess cell proliferation, 15 µM EdU was incorporated into the Caco-2 cells for 6 hours. Staining was performed using the Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 488 dye (Thermo Fisher Scientific), according to the manufacturer’s instructions. EdU staining was analyzed using CellProfiler 4.2.7 (Stirling et al., 2021 ) to quantify cell proliferation in Caco-2 cells cocultured with E. histolytica transformants. Images of EdU-labeled nuclei (Alexa Fluor™ 488) and Hoechst 33342-stained nuclei were processed to identify EdU-positive cells. Hoechst-stained nuclei were segmented as primary objects based on intensity thresholds, with object size constrained to 30–100 pixels to exclude artifacts. EdU-positive regions were identified as secondary objects using propagation-based segmentation linked to the primary nuclei. Illumination correction was applied to normalize intensity across EdU-stained images. Morphological measurements were extracted, including the proportion of EdU-positive cells relative to the total nuclei. Data from five images per condition were exported and statistically analyzed. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed using GraphPad Prism (version 10.2.0 MacOS, GraphPad Software, Boston, Massachusetts USA). Morphological Analysis Caco-2 cells cocultured with E. histolytica transformants were imaged as described in the IFA method above. Morphological analysis was conducted using CellProfiler 4.2.7. For each treatment, 5 images of Hoechst 33342-stained nuclei, E-cadherin-stained cell boundaries, and Phalloidin-stained F-actin were analyzed to quantify changes in cellular morphology. Nuclei were identified as primary objects using intensity-based segmentation of DNA-stained images, with a size range of 30–100 pixels. Cell boundaries were segmented as secondary objects using propagation-based methods, expanding from the nuclei and leveraging both E-cadherin and Phalloidin images for boundary definition. Illumination correction was applied to E-cadherin and Phalloidin images to normalize intensity variations. Data were exported as .csv files for statistical analysis, with parameters for segmentation and measurement optimized to ensure accurate object identification and quantification. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed using GraphPad Prism (version 10.2.0 MacOS, GraphPad Software). Morphological measurements were calculated, including: Form Factor, defined as \\(\\:\\text{Form:Factor}=\\frac{4{\\pi:}A}{{P}^{2}}\\) , where A is the area and P is the perimeter of the cell, used to assess circularity. Aspect Ratio, defined as \\(\\:\\text{Aspect:Ratio}=\\frac{\\text{Major:Axis}}{\\text{Minor:Axis}}\\) , representing the elongation of cells. Transepithelial Electrical Resistance (TEER) Measurement TEER was measured to assess epithelial barrier integrity using the EVOM™ Manual for TEER Measurement system (World Precision Instruments, Sarasota, FL, USA). Caco-2 cells were cultured on 12 mm Transwell inserts with a 0.4 µm pore size (3460, Corning Inc., Corning, NY, USA) as described in the cell culture method. Cells were maintained for 21 days until the TEER value stabilized at approximately 1300 Ω, indicating the formation of a confluent monolayer. Caco-2 cells were interacted with E. histolytica transformants at a ratio of 10:1 (Caco-2: E. histolytica ) or different concentration of recombinant proteins. TEER measurements were recorded before the interaction and at 1-hour intervals for up to 4 hours. Data at each time point were normalized to the baseline TEER value measured before interaction (time 0, set as 100%). Statistical significance at each time point was determined using a two-way ANOVA follwed by Dunnett’s multiple comparisons test on GraphPad Prism (version 10.2.0 MacOS, GraphPad Software). Wound Healing Assay Wound healing assay was conducted using Culture-Insert 2 Well in µ-Dish 35 mm (81176, ibidi GmbH, Gräfelfing, Germany) to create a Caco-2 monolayer with a defined gap. Caco-2 cells were cultured for 4 days to establish a confluent monolayer. E. histolytica transformants (2 × 10³) were seeded on either side of the insert and allowed to interact with the Caco-2 monolayer for 2 hours. Following interaction, E. histolytica was removed by washing with pre-cooled 2% galactose in PBS. To minimize the effects of cell proliferation, the Caco-2 monolayer was cultured in EMEM with 2% FBS after washing. Images of the gap area were captured at 1-hour intervals for up to 24 hours using an optical microscope, with 2–3 positions imaged per treatment group. Gap areas (width pixels) at each time point were measured using the Manual Wound Healing Size Tool plugin in Fiji software (Suarez-Arnedo et al., 2020 ). Recovery rates were determined by linear regression analysis of gap closure over time. Statistical significance of differences in recovery rates (slopes) between treatments was assessed using simple linear regression in GraphPad Prism (version 10.2.0 MacOS, GraphPad Software). Flow Cytometry Flow cytometry was performed to analyze Caco-2 cells treated with recombinant His-GFP-KERP2 or His-GFP proteins. Caco-2 cells were cultured for 21 days as described above and treated with 3 µM of His-GFP-KERP2 or His-GFP under various conditions. After treatment, cells were washed three times with 1× PBS and incubated with 0.5% trypsin for 5 minutes to remove excess recombinant protein and isolate the Caco-2 cells. The trypsinized cells were directly loaded into a BD Accuri™ C6 Plus flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) at a low flow rate. Data acquisition was performed using the instrument’s standard settings, and subsequent analysis and plotting were carried out using FlowJo software (BD Biosciences) and GraphPad Prism. Declarations Statistical information qRT-PCR: Statistical differences were determined by multiple unpaired t-tests. Growth kinetics: Statistical differences at individual time points were assessed using two-way ANOVA (n=3). Flow cytometry: Data acquisition was performed using a BD Accuri™ C6 Plus flow cytometer (BD Biosciences), and subsequent analysis of median fluorescence intensity and plotting were carried out using FlowJo software (BD Biosciences). Statistical differences of MFI at individual time points were assessed using two-way ANOVA (n=3). Cysteine protease (CP) activity assay: Statistical differences were evaluated using one-way ANOVA (n=8). EdU incorporation assay: Statistical significance for quantifying EdU-positive cells was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Morphological analysis: Form factor and aspect ratio were computed, and statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Wound healing assay: Recovery rates were determined by linear regression analysis of gap closure over time, and statistical significance of differences in recovery rates (slopes) between treatments was assessed using simple linear regression. Transepithelial Electrical Resistance (TEER) Measurement: Measurements were recorded at various time points, and statistical significance was determined using two-way ANOVA followed by Dunnett’s multiple comparisons test. Data availability RNA-seq data have been deposited at GEO: GSE290785 and GSE290901. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Acknowledgments This work was supported partly by Grants-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS) (JP21H02723 to T.N.), Fostering Joint International Research (B) from JSPS (JP21KK135 to T.N.), and Grant for Science and Technology Research Partnership for Sustainable Development (SATREPS) from Japan Agency for Medical Research and Development (AMED) and Japan International Cooperation Agency (JICA) (JP24jm0110022) to T.N., Grant for research on emerging and re-emerging infectious diseases from AMED (JP24fk0108680 to T.N.), and support from the University of Tokyo Pandemic Preparedness, Infection, and Advanced Research Center (UTOPIA) and AMED (JP243fa627001) to T.N.. This work was also partly supported by JSPS Grants-in-Aid for Scientific Research (23K06514 to H.J.S.), JSPS Bilateral Joint Research Grant (JPJSBP120223203 to H.J.S.)., and JST SPRING (Grant Number JPMJSP2108 to R.P.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank to Dr. Cecilia Villegas Novoa, Dr. Yuli Wang, and Prof. Nancy Allbritton from the Department of Bioengineering, University of Washington and Dr. Soichiro Ishihara and Dr. Yuzo Nagai from the Department of Surgery, University of Tokyo Hospital for their help in constructing 3D-crypt model. We thank to Dr. ISHINO Tomoko and Dr. Sinzawa Naoki from the Department of Parasitology and Tropical Medicine, Science Tokyo for their help in optimizing ChIP protocol. 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Cell Rep Methods 1 . 10.1016/j.crmeth.2021.100014 Zhou JK, Fan X, Cheng J, Liu W, Peng Y (2021) PDLIM1: Structure, function and implication in cancer. Cell Stress 5:119–127. 10.15698/cst2021.08.254 Zhou X, Zheng W, Li Y, Pearce R, Zhang C, Bell EW, Zhang G, Zhang Y (2022) I-TASSER-MTD: a deep-learning-based platform for multi-domain protein structure and function prediction. Nat Protoc 17:2326–2353. 10.1038/s41596-022-00728-0 Zuleger N, Robson MI, Schirmer EC (2011) The nuclear envelope as a chromatin organizer. Nucleus 2:339–349. 10.4161/nucl.2.5.17846 Zulfiqar H, Mathew G, Horrall S (2024) Amebiasis. In StatPearls Additional Declarations There is NO Competing Interest. Supplementary Files TableS1.xlsx Table S1. Primer list TableS2.xlsx Table S2. RNA-seq of KERPgs & psAP-mock TableS3.xlsx Table S3. E. histolytica HA-KERP2 co-IP proteome TableS4.xlsx Table S4. HA-KERP2 interactome in Caco-2 TableS5.xlsx Table S5. RNA-seq of Caco-2 under different conditions TableS6.xlsx Table S6. ChIP-seq for E. histolytica expressing HA-KERP2 VideoS1.mp4 Video S1. Live Imaging of GFP-HA-KERP2 Localization Time-lapse live imaging of GFP-HA-KERP2 during interaction. The GFP signal (green) indicates the localization of GFP-HA-KERP2, nuclei are stained with Hoechst 33342 (blue), and the merged image includes DIC to visualize cell morphology. VideoS2.mp4 Video S2. Live Imaging of GFP-HA-KERP2 ∆185−239 Localization Time-lapse live imaging of GFP-HA-KERP2 ∆185−239 during interaction. The GFP signal (green) indicates the localization of GFP-HA-KERP2 ∆185−239 , nuclei are stained with Hoechst 33342 (blue), and the merged image includes differential interference contrast (DIC) to visualize cell morphology. VideoS3.mp4 Video S3. Live Imaging of Caco-2 Interacting with GFP-HA-KERP2 Time-lapse live imaging of GFP-HA-KERP2 during interaction with Caco-2 cells. The GFP signal (green) represents GFP-HA-KERP2, while Hoechst 33342 (blue) stains the nuclei of Caco-2 cells. Green signals emerge progressively during the interaction, indicating the transfer of GFP-HA-KERP2. VideoS4.mp4 Video S4. Live Imaging of Caco-2 Interacting with GFP-HA-KERP2 Merged with DIC Time-lapse live imaging of GFP-HA-KERP2 interacting with Caco-2 cells, shown with merged DIC. GFP signal (green) represents GFP-HA-KERP2, and Hoechst 33342 (blue) stains the nuclei of Caco-2 cells. The DIC provides additional structural context, and green signals are observed appearing progressively during the interaction. DocumentS1.pdf Figure S1. Sequence Alignment and Structural Features of KERP2 (A) Sequence alignment of KERP2 (EHI_065630) with its homologs used for phylogenetic reconstruction. Conserved regions across Entamoeba species are highlighted, showing sequence similarities that define the KERP2 family. Only Entamoeba species are displayed due to the extensive list of homologs identified in broader eukaryotic taxa. (B) Alignment of KERP2-like DNA sequences identified in Polysphondylium pallidum and Dictyostelium purpureum. Weak conservation was observed in regions corresponding to the SAP domain. (C) Sequence alignment of the C-terminal regions of KERP2 and related DEK proteins, illustrating the lack of conservation in the C-terminal DNA-binding domain of KERP2. (D) Computational modeling of the surface charge distribution of KERP2 using APBS. The model highlights distinct regions with positive and negative charges. Figure S2. Validation of KERP2 Gene Silencing and Functional Assays (A) Electrophoresis analysis demonstrating KERP2 gene silencing in three independent E. histolytica psAP-KERP2gs transformants, validated by RT-PCR. RNA polymerase II was used as an endogenous control. (B-C) Growth kinetics of E. histolytica psAP-KERP2gs and psAP-mock transformants were compared in two additional independent trials, with measurements taken at 24-hour intervals over 96 hours. (D) Principal component analysis (PCA) of RNA-seq data, illustrating distinct transcriptomic profiles of psAP-KERP2gs and psAP-mock transformants. (E-H) Two additional independent trials comparing cysteine protease (CP) activities in intracellular (E, F) and released (G, H) forms among HA-KERP2_G3, HA-mock_G3, psAP-KERP2gs, and psAP-mock transformants. Figure S3. Analysis of HA-KERP2 Nuclear and Cytosolic Proteomes (A) Flamingo staining of nuclear and cytosolic co-IP pull-downs from HA-KERP2, HA-KERP2 ∆185−239 , and HA-Mock strains across four independent trials. Trial 4 was visualized using silver staining. (B-D) Western blot validation of nuclear and cytosolic co-IP pull-downs. (B) Trial 2, (C) Trial 3, and (D) Trial 4. Trial 1 is shown in the main Figure 2. Anti-CS1 and anti-Histone antibodies were used to validate successful nuclear and cytosolic fractionation. Anti-HA was used to detect the pull-down samples in the elution fractions. (E) KEGG pathway enrichment analysis of the HA-KERP2 nuclear proteome. (F) GO enrichment analysis of the HA-KERP2 nuclear proteome, highlighting molecular function categories. Figure S4. Negative Controls and Localization of KERP2 Variants (A) Live imaging of GFP-RtcB2 interacting with Caco-2 cells was conducted from 40 to 45 minutes. Differential interference contrast (DIC) images merged with the green channel (GFP-RtcB2) show interactions at the 40- and 45-minute time points. (B) Z-stack scanning was performed following live imaging. The green channel represents GFP-RtcB2, and the blue channel indicates Hoechst 33342-stained Caco-2 nuclei. (C) Western blot analysis of extracellular vesicle (EV) fractions isolated from HA-KERP2 and HA-KERP2 ∆185−239 . Immunoblot were detected using an anti-HA antibody. Figure S5. Purification and Validation of Recombinant His-GFP-KERP2 and His-GFP Proteins (A) Coomassie Brilliant Blue (CBB) staining of eluted recombinant His-GFP-KERP2 and His-GFP proteins, purified using an imidazole gradient after nickel-NTA pull-down. (B) Western blot analysis of recombinant proteins eluted with 750 mM imidazole, probed with anti-His antibody, confirming the presence of His-tagged His-GFP-KERP2 and His-GFP proteins. Figure S6. Transcriptome Analysis of Caco-2 Cells After Interaction with E. histolytica Strains (A) PCA showing the global transcriptome changes in Caco-2 cells after interaction with E. histolytica HA-KERP2-expressing G3 strain, KERP2gs knockdown strain, wild-type G3 strain, or untreated control. Distinct clusters represent transcriptional differences among treatment groups. (B) Volcano plot highlighting differentially expressed genes (DEGs) in Caco-2 cells interacting with HA-KERP2-expressing E. histolytica compared to untreated controls. Genes with adjusted p-value ≤ 0.05 and |log₂(fold change)| ≥ 1 are labeled as significantly upregulated or downregulated. (C) Volcano plot depicting DEGs in Caco-2 cells interacting with KERP2gs compared to untreated controls. (D) Volcano plot showing DEGs in Caco-2 cells interacting with wild-type G3 E. histolytica compared to untreated controls, representing baseline transcriptional changes induced by wild-type strain interaction. Figure S7. Morphological and Barrier Integrity Changes in Enterocytes and Caco-2 Cells After Interaction with E. histolytica Strains (A) Representative images of 3D-crypt enterocytes interacting with HA-KERP2 or KERP2gs. F-actin was stained with Phalloidin 594 (red), and nuclei were stained with Hoechst 33342 (blue). (B-E) TEER measurements in Caco-2 cells after treatment with HA-KERP2, HA-mock, KERP2gs, psAP-mock, or without treatment. (B, D) Line plots showing TEER changes over time for two independent trials, with points connected by lines. (C, E) Corresponding bar plots summarizing TEER values for each condition at different time points. (F-G) Two additional independent trials for dose-dependent effects of recombinant His-GFP-KERP2 on TEER reduction. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-6191032\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":436851640,\"identity\":\"5e2b4cca-c43f-4751-8bdf-693626bde92a\",\"order_by\":0,\"name\":\"Tomoyoshi Nozaki\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYJACxgYDGyjzAJSWIKwlDUKToIXhMJoWfMCcvf3hxxkF56MZJBLYH3w4c1iOgf3wAwbLHbi1WPYcSJbcYHA7t0EigbFxxo3Dxgw8aQYMkmdwazG4kXBA8gFYS/7HZp4PhxMbGHIYGCTb8GlJbP75wOAc2BaIFv43hLQkswEddgCq5QZQiwQhW84cY7OcYZCc28bzgHHmjDPpxmwSzwwO4PXL8fbHN3v+2OX2sycwfPhwzFqOnz/54WNJPCEGB2wQqhnMOCzZQIQWKKgDk4wfSdAyCkbBKBgFwx4AADEvVN1f7uuYAAAAAElFTkSuQmCC\",\"orcid\":\"https://orcid.org/0000-0003-1354-5133\",\"institution\":\"The University of Tokyo\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Tomoyoshi\",\"middleName\":\"\",\"lastName\":\"Nozaki\",\"suffix\":\"\"},{\"id\":436851641,\"identity\":\"85970e5f-6b6d-4c89-af05-b4c95f19bbea\",\"order_by\":1,\"name\":\"Ruofan Peng\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0002-1441-3741\",\"institution\":\"The University of Tokyo\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ruofan\",\"middleName\":\"\",\"lastName\":\"Peng\",\"suffix\":\"\"},{\"id\":436851642,\"identity\":\"4872953d-3392-4039-83de-957f9e1cfed7\",\"order_by\":2,\"name\":\"Herbert Santos\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"The University of Tokyo\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Herbert\",\"middleName\":\"\",\"lastName\":\"Santos\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-03-10 02:05:20\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6191032/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6191032/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":81510409,\"identity\":\"fc4b2508-a47c-40b3-ad23-6e6e06e5226c\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:53\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":174777,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eStructural and Functional Characterization of KERP2 through In Silico Analysis.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Maximum likelihood phylogenetic tree of KERP2 (EHI_065630) and homologous sequences across eukaryotic taxa, constructed using IQ-TREE and visualized with iTOL. The circular display represents bootstrap values with color gradients, while the rectangular display provides a simplified view for clarity, focusing on \\u003cem\\u003eEntamoeba\\u003c/em\\u003especies. Bootstrap values are indicated at major branch points.\\u003cbr\\u003e\\n(B) Multiple sequence alignment of the KERP2 SAP domain (amino acids 104-141) with representative SAP domains from other organisms, constructed as described in (Aravind and Koonin, 2000). Conserved residues associated with DNA binding are highlighted by residue properties: hydrophobic (yellow), small (green), polar (purple), and bulky (gray). Predicted secondary structures, shown schematically, feature two amphipathic helices characteristic of the SAP domain. \\u003cbr\\u003e\\n(C) Structural model of KERP2 generated by I-TASSER (white) showing conserved architecture resembling the N-terminal region of DEK proteins, including the embedded SAP domain (green; PDB ID: 2JX3). \\u003cbr\\u003e\\n(D) Sequence analysis identifies a coiled-coil domain in the C-terminal region (amino acids 178–216) predicted by PCOILS and MARCOIL. \\u003cbr\\u003e\\n(E) Schematic representation of the domain architecture of KERP2, highlighting the SAP domain (green), coiled-coil domain (purple), and nuclear localization signal (NLS, red line).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/34accc26a7e5a740ba2c910d.jpg\"},{\"id\":81510306,\"identity\":\"0b66bb30-0f90-4c47-a8b1-8e0325a0b91a\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:48\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":150510,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eIntracellular localization and subcellular distribution of KERP2 variants in E. histolytica.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Schematic representation of HA-KERP2 constructs used in this study: full-length HA-KERP2 and truncated HA-KERP2\\u003csup\\u003e∆185−239\\u003c/sup\\u003e lacking the coiled-coil domain.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) IFA of\\u0026nbsp;\\u003cem\\u003eE. histolytica\\u003c/em\\u003e\\u0026nbsp;trophozoites expressing HA-KERP2 constructs. Cells were stained with anti-HA (green), anti-cysteine synthase 1 (red) as a cytosol marker, and Hoechst 33342 (blue) to visualize nuclei. White arrowheads indicate the nuclear localization, while white arrows highlight cytosolic puncta or vesicular structures. Line intensity profile analysis demonstrates colocalization across channels.\\u003c/p\\u003e\\n\\u003cp\\u003e(C–D) Immuno-EM analysis of \\u003cem\\u003eE. histolytica\\u003c/em\\u003e trophozoites expressing HA-KERP2. (C) HA-KERP2 is enriched in chromatin-dense regions (white arrows). Light blue circles highlight HA-KERP2 signals within the nucleus, while yellow circles indicate signals in the cytosol. (D) HA-KERP2 is detected in small electron-dense granule (EDG)-like structures, the cytosol, and large vesicles (white arrows). Yellow circles highlight HA-KERP2 signals.\\u003c/p\\u003e\\n\\u003cp\\u003e(E) Subcellular fractionation of\\u0026nbsp;\\u003cem\\u003eE. histolytica\\u003c/em\\u003e\\u0026nbsp;expressing HA-KERP2, HA-KERP2\\u003csup\\u003e∆185−239\\u003c/sup\\u003e, and HA-mock constructs. Nuclear/membrane components (14,000G pellet) and cytosolic/vesicular components (supernatant) were separated and validated by Western blot using anti-cysteine synthase 1 (cytosol marker) and anti-histone (nuclear marker).\\u003c/p\\u003e\\n\\u003cp\\u003e(F) HA pull-down from fractionated samples followed by Western blot analysis using anti-HA antibody to detect HA-KERP2 and HA-KERP2\\u003csup\\u003e∆185−239\\u003c/sup\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/2051e04e3ce032d833415cbe.jpg\"},{\"id\":81510368,\"identity\":\"7d9af5a3-ad55-4345-a719-7443ac5755ba\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:52\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":129637,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eGene silencing of KERP2 and its impact on E. histolytica metabolism and virulence.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Quantitative reverse transcription PCR (qRT-PCR) analysis confirming efficient knockdown of KERP2 in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e. Three independent KERP2-knockdown strains (psAP-KERP2gs) compared to their psAP-mock controls are shown. RNA polymerase II was used as an endogenous control. Statistical differences were determined by multiple unpaired t-tests.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Growth curve analysis showing no significant differences in proliferation between psAP-KERP2gs and psAP-mock strains under the tested conditions. Growth was monitored at 24-hour intervals up to 96 hours. \\u003cem\\u003en\\u003c/em\\u003e = 3; statistical differences at individual time points were assessed using two-way ANOVA.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) GO and KEGG pathway enrichment analyses of RNA-Seq data from psAP-KERP2gs. Enrichment highlights significant upregulation of genes related to proteolysis, cysteine metabolism, and amoebiasis.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Differential expression of key genes in psAP-KERP2gs, including upregulation of cysteine synthases, cysteine protease, and amoebapore precursors.\\u003c/p\\u003e\\n\\u003cp\\u003e(E, F) Intracellular (E) and released (F) CP activity was measured and expressed as fluorescence intensity corresponding to the production of 4-methylcoumaryl-7-amide per milligram of lysate protein. Statistical significance was assessed using one-way ANOVA. Data are presented as the mean ± standard deviation (n = 8).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/b2ca8a9633ab5a3ed6333ab2.jpg\"},{\"id\":81510220,\"identity\":\"bddb52e5-d6c6-43c9-bb5d-e2e75d2f2331\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:43\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":129922,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNuclear co-immunoprecipitation and protein-protein interactions of HA-KERP2 in E. histolytica.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Venn diagram showing significant interactors from HA-KERP2 nuclear proteome identified through co-IP and MS analysis. Proteins were classified as double or triple hits based on quantitative values (QV) compared to HA-mock controls across replicates with adjusted p-value \\u0026lt; 0.05.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Venn diagram of HA-KERP2 cytosolic proteome, highlighting double and triple hits as described above.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Enrichment analysis of HA-KERP2 nuclear interactome showing associations with cellular components such as vesicles, non-membrane-bounded organelles, and ribosomes.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) KEGG pathway enrichment analysis of HA-KERP2 cytosolic interactome.\\u003c/p\\u003e\\n\\u003cp\\u003e(E) Heat map displaying QV comparison of proteins from HA-KERP2, HA-KERP2\\u003csup\\u003e∆185−239\\u003c/sup\\u003e, and HA-mock nuclear interactome, grouped into functional categories: protein transport, protein folding, nucleic acid binding, and ribosome biogenesis.\\u003c/p\\u003e\\n\\u003cp\\u003e(F) Heat map of cytosolic interactome is displayed same as above and grouped into functional categories: protein transport, protein folding, and nucleic acid binding.\\u003c/p\\u003e\\n\\u003cp\\u003e(G) IFA of \\u003cem\\u003eE. histolytica\\u003c/em\\u003e trophozoites stained with anti-Rab11B (red) and anti-HA (green). Co-localization of HA-KERP2 and Rab11B is highlighted by white arrows, while white arrowheads indicate nuclear localization of HA-KERP2.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/a423d1d01614aa7ed78d99a5.jpg\"},{\"id\":81510410,\"identity\":\"89324238-fdaa-4746-9383-283bbae0ffa9\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:53\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":138870,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eKERP2 translocation from E. histolytica to host epithelial cells\\u003c/p\\u003e\\n\\u003cp\\u003e(A) IFA images of Caco-2 cells co-cultured with HA-KERP2-overexpressing \\u003cem\\u003eE. histolytica\\u003c/em\\u003e trophozoites for 2 hours, showing weak punctate HA-KERP2 signals (green) in the cytoplasm and partial nuclear localization in Caco-2 cells. Nuclei were stained with Hoechst 33342 (blue), and F-actin was labeled with Alexa Fluor 594 Phalloidin (red). The upper panel displays a single optical plane, while the lower panel shows a 3D projection generated from Z-stack scanning.\\u003cbr\\u003e\\n(B-C) Live imaging of GFP-HA-KERP2-expressing \\u003cem\\u003eE. histolytica\\u003c/em\\u003e interacting with a Caco-2 monolayer. (B) DIC merged images showing the interaction after 30 and 45 minutes. (C) Z-stack images of Zone 1 captured after the video recording. Green signals represent GFP-HA-KERP2, and blue signals indicate Hoechst 33342-stained Caco-2 nuclei.\\u003c/p\\u003e\\n\\u003cp\\u003e(D-E) A 3D intestinal crypt model was used to validate KERP2 translocation under physiological conditions. (D) Schematic illustration of the 3D crypt model and (E) IFA images of differentiated enterocytes after 2 hours of interaction with GFP-HA-KERP2-expressing or GFP-HA \\u003cem\\u003eE. histolytica\\u003c/em\\u003e. The crypts were stained with HA antibody (green), Hoechst 33342 (blue), and CellTracker Deep Red, specific to \\u003cem\\u003eE. histolytica\\u003c/em\\u003e(magenta). \\u003cbr\\u003e\\n(F) Western blot analysis of Caco-2 and \\u003cem\\u003eE. histolytica\\u003c/em\\u003e lysates following co-culture for 1, 2, and 3 hours confirmed the presence of HA-KERP2 (~35 kDa indicated by arrow) in Caco-2 cells, with translocation increasing over time. Treatment with 2% galactose significantly reduced KERP2 translocation. Successful separation of trophozoites and Caco-2 cells was verified using CS-1 (specific for \\u003cem\\u003eE. histolytica\\u003c/em\\u003e) and GAPDH (specific for Caco-2 cells) antibodies.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/0630a0ee62c1883c14073bc8.jpg\"},{\"id\":81510329,\"identity\":\"c9fba199-7926-4fae-af46-ff610073e596\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:49\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":158389,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eKERP2 Trafficking Mechanism in E. histolytica and Caco-2 Cells.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Immuno-EM analysis of HA-KERP2 localization in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e and Caco-2 cells after 1-hour co-culture. The first images display an overview, with the orange and green boxes highlighting regions that are shown in higher magnification on the right. HA-KERP2 signals in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e are circled in yellow, while those in Caco-2 cells are circled in light blue. Plasma membrane, microvilli, granule-like structures, and endosome-like structures are indicated by black arrows.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Representative FACS plots showing forward scatter area (FSC-A) versus fluorescence intensity (FITC-A) for Caco-2 cells trypsinized after a 1-hour incubation with 3 μM His-GFP-KERP2 (upper panel) or His-GFP (lower panel).\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Median fluorescence intensity (MFI) of GFP measured over a 24-hour period for Caco-2 cells treated with His-GFP-KERP2 or His-GFP.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) FACS plots of Caco-2 cells incubated with His-GFP-KERP2 or RITC-dextran for 4 hours at 35.5°C or 4°C.\\u003c/p\\u003e\\n\\u003cp\\u003e(E) FACS plots of Caco-2 cells exposed to His-GFP-KERP2 or His-GFP for 24 hours, washed, and analyzed at 24 hours (post-24 h) and 48 hours (post-48 h) post-wash.\\u003c/p\\u003e\\n\\u003cp\\u003e(F) Lipid overlay assay of recombinant His-KERP2 and His-GFP (negative control). Membrane strips contain 100 pmol of each lipid per spot, including lysophosphatidic acid (LPA), lysophosphocholine (LPC), phosphatidylinositol (PtdIns), phosphatidylinositol (3)-phosphate [PI(3)P], phosphatidylinositol (4)-phosphate [PI(4)P], phosphatidylinositol (5)-phosphate [PI(5)P], phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingosine 1-phosphate (S1P), phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2], phosphatidylinositol (3,5)-bisphosphate [PI(3,5)P2], phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2], phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], phosphatidic acid (PA), and phosphatidylserine (PS).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/2fdeaf03a5c38398ccced663.jpg\"},{\"id\":81510244,\"identity\":\"6312ab38-7b9c-4c90-9e39-1d1ca4d5f347\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:44\",\"extension\":\"jpg\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":120780,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePhysiological effects of KERP2 on host epithelial cells.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Co-IP followed by MS analysis of HA-KERP2 interactors in Caco-2 cells. Venn diagram shows significant interactors categorized as double or triple hits, defined by at least two-fold higher abundance in HA-KERP2 samples compared to HA-mock controls, with an adjusted p-value \\u0026lt; 0.05 across replicates.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) GO enrichment analysis of the KERP2 interactome, highlighting key functional categories such as cadherin binding, actin binding, and transcription coactivator binding.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Functional classification of KERP2 interactors in Caco-2 cells, with corresponding quantitative value (QV) comparisons. Categories include cell cycle regulation, cytoskeletal dynamics, cell-cell adhesion (e.g., CTNNB1, ITGB1), intracellular traffickin, and immune responses, and gene regulation.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) PCA of RNA-seq data from Caco-2 cells co-cultured with HA-KERP2-overexpressing, psAP-KERP2gs, or wild-type \\u003cem\\u003eE. histolytica\\u003c/em\\u003estrains, demonstrating distinct clustering of gene expression profiles.\\u003c/p\\u003e\\n\\u003cp\\u003e(E) Scatter plot showing genes with two-fold changes in expression in one strain but not the other. Log2(fold change) values for HA-KERP2 (x-axis) and psAP-KERP2gs (y-axis) strains are plotted. Significant genes (adjusted p-value \\u0026lt; 0.05) are highlighted: red for genes significantly upregulated in HA-KERP2, blue for genes significantly upregulated in psAP-KERP2gs, and green for genes with significant changes in both strains.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/2588c606f978c94aa9e80fcc.jpg\"},{\"id\":81510419,\"identity\":\"dbacef82-2c2f-4cb2-a198-d2a6e5c40a38\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:53\",\"extension\":\"jpg\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":201197,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFunctional analysis of KERP2 effects on Caco-2 host cells.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Confocal microscopy images showing EdU incorporation in Caco-2 cells co-cultured with or without \\u003cem\\u003eE. histolytica\\u003c/em\\u003e strains HA-KERP2, HA-mock, KERP2gs, or psAP-mock for 2 hours, followed by 6 hours of EdU labeling. EdU-positive nuclei (green) were visualized using the Click-iT kit, and Hoechst 33342 (blue) was used to stain all nuclei.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Quantification of EdU-positive nuclei. Data were derived from five input images per condition and analyzed for statistical significance using one-way ANOVA.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Immunofluorescence staining for apical view of Caco-2 cells co-cultured with \\u003cem\\u003eE. histolytica\\u003c/em\\u003e strains after 2 hours. Hoechst 33342 (blue) labels nuclei, anti-E-cadherin (green) marks tight junctions, and phalloidin (red) stains F-actin.\\u003c/p\\u003e\\n\\u003cp\\u003e(D, E) Quantification of cell morphology. (D) Form factor of Caco-2 cells was calculated following treatment with HA-KERP2, KERP2gs, or no treatment (control). (E) Aspect ratio was similarly calculated. The equations used for these measurements are detailed in the Methods section. Data were derived from five input images per condition and analyzed for statistical significance using one-way ANOVA.\\u003c/p\\u003e\\n\\u003cp\\u003e(F) Immunofluorescence staining for basal view of Caco-2 cells co-cultured with \\u003cem\\u003eE. histolytica\\u003c/em\\u003e strains after 2 hours. Phalloidin in red stains F-actin.\\u003c/p\\u003e\\n\\u003cp\\u003e(G) Representative microscope images of wound healing assay tracking cell motility after co-culture with \\u003cem\\u003eE. histolytica\\u003c/em\\u003e strains HA-KERP2, HA-mock, KERP2gs, or psAP-mock at time 0 and time 19 hours.\\u003c/p\\u003e\\n\\u003cp\\u003e(H) Linear regression analysis of wound closure rates was performed. Two images were analyzed for each condition. The gap area was measured at each time point to determine wound healing rates. Differences in recovery rates (slopes) between treatments were evaluated for statistical significance using simple linear regression.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"8.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/e5aeb3c715f9320176a66f51.jpg\"},{\"id\":81510372,\"identity\":\"3bc0acff-86d0-4810-b588-cc0b0a5bade0\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:52\",\"extension\":\"jpg\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":80699,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFunctional analysis of KERP2 effects on Caco-2 host cells.\\u003c/p\\u003e\\n\\u003cp\\u003e(A-C) TEER measurements of Caco-2 cells treated with \\u003cem\\u003eE. histolytica\\u003c/em\\u003e strains HA-KERP2, HA-mock, KERP2gs, psAP-mock, and untreated controls. (A) Line plot of TEER over time (n = 3). (B) Bar plot summarizing TEER values. (C) Data values corresponding to (H) and (I). Statistical significance at different time points was determined using two-way ANOVA.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Line plot of TEER measurements (n=3) following treatment with recombinant His-GFP-KERP2 (1.5 µM, 3 µM, 6 µM) and His-GFP (3 µM, 6 µM) at increasing concentrations.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"9.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/f787e9731897add988cff55a.jpg\"},{\"id\":81510301,\"identity\":\"7c06919b-a9b4-4755-a970-9e03ffd63f8b\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:47\",\"extension\":\"xlsx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":10929,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eTable S1. Primer list\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"TableS1.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/81c244824897a52a66483afa.xlsx\"},{\"id\":81510469,\"identity\":\"c601298d-e6a2-4dae-ad03-0c46f674a27d\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:56\",\"extension\":\"xlsx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1018283,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eTable S2. RNA-seq of KERPgs \\u0026amp; psAP-mock\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"TableS2.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/1423c0eb8af80410a7799595.xlsx\"},{\"id\":81510349,\"identity\":\"12c1ed4a-2a7a-4472-a7f1-62592cceda21\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:50\",\"extension\":\"xlsx\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":245081,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eTable S3. \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eE. histolytica\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e HA-KERP2 co-IP proteome\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"TableS3.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/dbafdaade96d39c343a8587a.xlsx\"},{\"id\":81510336,\"identity\":\"53a2c434-b253-44ad-84a5-62668d0c63b6\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:49\",\"extension\":\"xlsx\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":362861,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eTable S4. HA-KERP2 interactome in Caco-2\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"TableS4.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/f16f6a896973347f930c6117.xlsx\"},{\"id\":81511214,\"identity\":\"19223ed9-336b-4dac-9ccc-8b59bb7ef7e4\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:12:51\",\"extension\":\"xlsx\",\"order_by\":5,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":5652711,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eTable S5. RNA-seq of Caco-2 under different conditions\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"TableS5.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/e0f692e06fd3d04a8481f37b.xlsx\"},{\"id\":81510311,\"identity\":\"1f8c4cf1-bd76-462c-873d-5d529d252853\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:48\",\"extension\":\"xlsx\",\"order_by\":6,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":587741,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eTable S6. ChIP-seq for \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eE. histolytica\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e expressing HA-KERP2\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"TableS6.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/07a1774299b2bde936732f6b.xlsx\"},{\"id\":81510298,\"identity\":\"24200488-bdb7-48b3-bde3-3ba407af4d27\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:47\",\"extension\":\"mp4\",\"order_by\":7,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1981256,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eVideo S1. Live Imaging of GFP-HA-KERP2 Localization\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTime-lapse live imaging of GFP-HA-KERP2 during interaction. The GFP signal (green) indicates the localization of GFP-HA-KERP2, nuclei are stained with Hoechst 33342 (blue), and the merged image includes DIC to visualize cell morphology.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"VideoS1.mp4\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/de0fad3a6450218a2e7d1c1f.mp4\"},{\"id\":81510305,\"identity\":\"1dfa3288-7b48-40c2-b926-f31dc2a6c361\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:48\",\"extension\":\"mp4\",\"order_by\":8,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":3540447,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eVideo S2. Live Imaging of GFP-HA-KERP2\\u003c/strong\\u003e\\u003csup\\u003e\\u003cstrong\\u003e∆185−239\\u003c/strong\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003e Localization\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTime-lapse live imaging of GFP-HA-KERP2\\u003csup\\u003e∆185−239\\u003c/sup\\u003e during interaction. The GFP signal (green) indicates the localization of GFP-HA-KERP2\\u003csup\\u003e∆185−239\\u003c/sup\\u003e, nuclei are stained with Hoechst 33342 (blue), and the merged image includes differential interference contrast (DIC) to visualize cell morphology.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"VideoS2.mp4\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/ecb4e1f76bdcfc5afae19b83.mp4\"},{\"id\":81510315,\"identity\":\"b4a2460e-21e6-478c-965e-69c52a909e05\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:48\",\"extension\":\"mp4\",\"order_by\":9,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":25102881,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eVideo S3. Live Imaging of Caco-2 Interacting with GFP-HA-KERP2\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTime-lapse live imaging of GFP-HA-KERP2 during interaction with Caco-2 cells. The GFP signal (green) represents GFP-HA-KERP2, while Hoechst 33342 (blue) stains the nuclei of Caco-2 cells. Green signals emerge progressively during the interaction, indicating the transfer of GFP-HA-KERP2.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"VideoS3.mp4\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/68b594977ff861995415abce.mp4\"},{\"id\":81510362,\"identity\":\"aacf5720-5d07-4b2f-8a59-00632a27d424\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:51\",\"extension\":\"mp4\",\"order_by\":10,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":82544388,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eVideo S4. Live Imaging of Caco-2 Interacting with GFP-HA-KERP2 Merged with DIC\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTime-lapse live imaging of GFP-HA-KERP2 interacting with Caco-2 cells, shown with merged DIC. GFP signal (green) represents GFP-HA-KERP2, and Hoechst 33342 (blue) stains the nuclei of Caco-2 cells. The DIC provides additional structural context, and green signals are observed appearing progressively during the interaction.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"VideoS4.mp4\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/59bc245d2c17de27b16a9a78.mp4\"},{\"id\":81510354,\"identity\":\"aa19d786-e3e4-4c34-8426-27035885600f\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 06:04:51\",\"extension\":\"pdf\",\"order_by\":11,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":91130134,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFigure S1. Sequence Alignment and Structural Features of KERP2\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Sequence alignment of KERP2 (EHI_065630) with its homologs used for phylogenetic reconstruction. Conserved regions across Entamoeba species are highlighted, showing sequence similarities that define the KERP2 family. Only Entamoeba species are displayed due to the extensive list of homologs identified in broader eukaryotic taxa.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Alignment of KERP2-like DNA sequences identified in Polysphondylium pallidum and Dictyostelium purpureum. Weak conservation was observed in regions corresponding to the SAP domain.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Sequence alignment of the C-terminal regions of KERP2 and related DEK proteins, illustrating the lack of conservation in the C-terminal DNA-binding domain of KERP2.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Computational modeling of the surface charge distribution of KERP2 using APBS. The model highlights distinct regions with positive and negative charges.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;Figure S2. Validation of KERP2 Gene Silencing and Functional Assays\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Electrophoresis analysis demonstrating KERP2 gene silencing in three independent E. histolytica psAP-KERP2gs transformants, validated by RT-PCR. RNA polymerase II was used as an endogenous control.\\u003c/p\\u003e\\n\\u003cp\\u003e(B-C) Growth kinetics of E. histolytica psAP-KERP2gs and psAP-mock transformants were compared in two additional independent trials, with measurements taken at 24-hour intervals over 96 hours.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Principal component analysis (PCA) of RNA-seq data, illustrating distinct transcriptomic profiles of psAP-KERP2gs and psAP-mock transformants.\\u003c/p\\u003e\\n\\u003cp\\u003e(E-H) Two additional independent trials comparing cysteine protease (CP) activities in intracellular (E, F) and released (G, H) forms among HA-KERP2_G3, HA-mock_G3, psAP-KERP2gs, and psAP-mock transformants.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure S3. Analysis of HA-KERP2 Nuclear and Cytosolic Proteomes\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Flamingo staining of nuclear and cytosolic co-IP pull-downs from HA-KERP2, HA-KERP2\\u003csup\\u003e∆185−239\\u003c/sup\\u003e, and HA-Mock strains across four independent trials. Trial 4 was visualized using silver staining.\\u003c/p\\u003e\\n\\u003cp\\u003e(B-D) Western blot validation of nuclear and cytosolic co-IP pull-downs. (B) Trial 2, (C) Trial 3, and (D) Trial 4. Trial 1 is shown in the main Figure 2. Anti-CS1 and anti-Histone antibodies were used to validate successful nuclear and cytosolic fractionation. Anti-HA was used to detect the pull-down samples in the elution fractions.\\u003c/p\\u003e\\n\\u003cp\\u003e(E) KEGG pathway enrichment analysis of the HA-KERP2 nuclear proteome.\\u003c/p\\u003e\\n\\u003cp\\u003e(F) GO enrichment analysis of the HA-KERP2 nuclear proteome, highlighting molecular function categories.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003cstrong\\u003eFigure S4. Negative Controls and Localization of KERP2 Variants\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Live imaging of GFP-RtcB2 interacting with Caco-2 cells was conducted from 40 to 45 minutes. Differential interference contrast (DIC) images merged with the green channel (GFP-RtcB2) show interactions at the 40- and 45-minute time points.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Z-stack scanning was performed following live imaging. The green channel represents GFP-RtcB2, and the blue channel indicates Hoechst 33342-stained Caco-2 nuclei.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Western blot analysis of extracellular vesicle (EV) fractions isolated from HA-KERP2 and HA-KERP2\\u003csup\\u003e∆185−239\\u003c/sup\\u003e. Immunoblot were detected using an anti-HA antibody.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure S5. Purification and Validation of Recombinant His-GFP-KERP2 and His-GFP Proteins\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Coomassie Brilliant Blue (CBB) staining of eluted recombinant His-GFP-KERP2 and His-GFP proteins, purified using an imidazole gradient after nickel-NTA pull-down.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Western blot analysis of recombinant proteins eluted with 750 mM imidazole, probed with anti-His antibody, confirming the presence of His-tagged His-GFP-KERP2 and His-GFP proteins.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure S6. Transcriptome Analysis of Caco-2 Cells After Interaction with E. histolytica Strains\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) PCA showing the global transcriptome changes in Caco-2 cells after interaction with E. histolytica HA-KERP2-expressing G3 strain, KERP2gs knockdown strain, wild-type G3 strain, or untreated control. Distinct clusters represent transcriptional differences among treatment groups.\\u003c/p\\u003e\\n\\u003cp\\u003e(B) Volcano plot highlighting differentially expressed genes (DEGs) in Caco-2 cells interacting with HA-KERP2-expressing E. histolytica compared to untreated controls. Genes with adjusted p-value ≤ 0.05 and |log₂(fold change)| ≥ 1 are labeled as significantly upregulated or downregulated.\\u003c/p\\u003e\\n\\u003cp\\u003e(C) Volcano plot depicting DEGs in Caco-2 cells interacting with KERP2gs compared to untreated controls.\\u003c/p\\u003e\\n\\u003cp\\u003e(D) Volcano plot showing DEGs in Caco-2 cells interacting with wild-type G3 E. histolytica compared to untreated controls, representing baseline transcriptional changes induced by wild-type strain interaction.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure S7. Morphological and Barrier Integrity Changes in Enterocytes and Caco-2 Cells After Interaction with E. histolytica Strains\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Representative images of 3D-crypt enterocytes interacting with HA-KERP2 or KERP2gs. F-actin was stained with Phalloidin 594 (red), and nuclei were stained with Hoechst 33342 (blue).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;(B-E) TEER measurements in Caco-2 cells after treatment with HA-KERP2, HA-mock, KERP2gs, psAP-mock, or without treatment. (B, D) Line plots showing TEER changes over time for two independent trials, with points connected by lines. (C, E) Corresponding bar plots summarizing TEER values for each condition at different time points.\\u003c/p\\u003e\\n\\u003cp\\u003e(F-G) Two additional independent trials for dose-dependent effects of recombinant His-GFP-KERP2 on TEER reduction.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"DocumentS1.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6191032/v1/851504232f5b1509f87f7523.pdf\"}],\"financialInterests\":\"There is \\u003cb\\u003eNO\\u003c/b\\u003e Competing Interest.\",\"formattedTitle\":\"Dual Role of Entamoeba histolytica KERP2 in Regulating Gene Expression and Modulating Host Cell Function for Intestinal Colonization\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003ePathogens have evolved sophisticated mechanisms to hijack host cellular functions, enabling their survival, replication, and transmission. Unlike commensal microbes, which coexist with the host without triggering strong immune responses, pathogenic microbes actively manipulate host cells to evade immune detection and create environments favorable for infection (Arrieta and Finlay, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e; Littman and Pamer, \\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e; Tanoue et al., \\u003cspan citationid=\\\"CR99\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e). Intracellular pathogens, such as \\u003cem\\u003eMycobacterium tuberculosis\\u003c/em\\u003e, \\u003cem\\u003eListeria monocytogenes\\u003c/em\\u003e, \\u003cem\\u003ePlasmodium falciparum\\u003c/em\\u003e, and \\u003cem\\u003eToxoplasma gondii\\u003c/em\\u003e, invade host cells to escape immune surveillance and establish specialized intracellular niches (Disson et al., \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e; Koch and Mizrahi, \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Lourido, \\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Maier et al., \\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). These pathogens subvert host cellular machinery to facilitate their replication by modifying endocytic trafficking, preventing lysosomal fusion, or hijacking host cytoskeletal components for movement and dissemination. In contrast, extracellular pathogens remain outside host cells but still engage in complex interactions with host tissues to establish infection and evade immune responses. Many extracellular bacteria, such as \\u003cem\\u003eVibrio cholerae\\u003c/em\\u003e, \\u003cem\\u003eStreptococcus pneumoniae\\u003c/em\\u003e, and \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e, produce virulence factors like toxins or adhesins that modulate host signaling pathways and immune responses (Cho et al., \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e; Howden et al., \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Weiser et al., \\u003cspan citationid=\\\"CR105\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Similarly, extracellular parasites, including \\u003cem\\u003eTrypanosoma brucei\\u003c/em\\u003e, \\u003cem\\u003eGiardia lamblia\\u003c/em\\u003e, and \\u003cem\\u003eEntamoeba histolytica\\u003c/em\\u003e, have evolved intricate strategies to manipulate host cell functions despite not residing within them (Begum et al., \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e; Einarsson et al., \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Romero-Meza and Mugnier, \\u003cspan citationid=\\\"CR80\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eExtracellular parasites exploit host cell surfaces, secreted factors, and molecular mimicry to ensure their survival and persistence. Some, like \\u003cem\\u003eT. brucei\\u003c/em\\u003e, evade immune recognition by continuously altering their surface glycoproteins (variant surface glycoproteins, VSGs) through antigenic variation (Mugnier et al., \\u003cspan citationid=\\\"CR69\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Mugnier et al., \\u003cspan citationid=\\\"CR70\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). Others, such as \\u003cem\\u003eGiardia\\u003c/em\\u003e, disrupt host epithelial barrier integrity by attaching to the intestinal epithelium via its ventral adhesive disc, a microtubule-based structure (Lanfredi-Rangel et al., \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e1999\\u003c/span\\u003e; Schwartz et al., \\u003cspan citationid=\\\"CR88\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). This attachment induces mechanical stress, disrupting tight junction proteins such as claudin-1, occludin, and ZO-1, ultimately increasing intestinal permeability (Buret et al., \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2002\\u003c/span\\u003e; Maia-Brigagao et al., \\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). Additionally, \\u003cem\\u003eGiardia\\u003c/em\\u003e also secretes cysteine proteases that degrade host mucins and tight junction proteins, exacerbating epithelial damage and facilitating nutrient uptake (Bhargava et al., \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Liu et al., \\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eAmong extracellular protozoan parasites, \\u003cem\\u003eE. histolytica\\u003c/em\\u003e is particularly notable for its dual role as both a commensal and a pathogen in the human gastrointestinal tract. \\u003cem\\u003eE. histolytica\\u003c/em\\u003e is responsible for amoebiasis, a disease of significant global health concern that affects up to 50\\u0026nbsp;million people annually, causing over 70,000 deaths worldwide (Bercu et al., \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e; Kantor et al., \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Ximenez et al., \\u003cspan citationid=\\\"CR110\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e). Infections often begin asymptomatically but can progress to severe symptoms, including abdominal pain, watery or bloody diarrhea, and, in some cases, life-threatening complications such as liver abscesses, pneumonia, purulent pericarditis, and cerebral amoebiasis (El-Dib, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Kantor et al., \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Sharma and Ahuja, \\u003cspan citationid=\\\"CR90\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e). Within the intestine, \\u003cem\\u003eE. histolytica\\u003c/em\\u003e induces severe inflammation, tissue perforation, and fulminant amoebic colitis, leading to potentially fatal outcomes (Shirley et al., \\u003cspan citationid=\\\"CR91\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Zulfiqar et al., \\u003cspan citationid=\\\"CR118\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Although infection rates are declining in many low and middle income countries, an increase has been observed in high-income East Asian regions including Japan and Taiwan and in parts of Europe over the past two decades (Fu et al., \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Lin et al., \\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Yanagawa et al., \\u003cspan citationid=\\\"CR112\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Populations at high risk include residents of endemic regions (e.g., Mexico, Central and South America, Asia, Africa, and the Pacific Islands), immigrants and travelers from these areas, men who have sex with men (MSM), and people living or working in group facilities (Ansart et al., \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e; Hung et al., \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e; Stanley, \\u003cspan citationid=\\\"CR96\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Weinke et al., \\u003cspan citationid=\\\"CR104\\\" class=\\\"CitationRef\\\"\\u003e1990\\u003c/span\\u003e). The coexistance of asymptomatic carriers and symptomatic individuals highlights \\u003cem\\u003eE. histolytica\\u0026rsquo;s\\u003c/em\\u003e potential for widespread transmission and underscores the critical need to elucidate its pathogenic mechanisms.\\u003c/p\\u003e \\u003cp\\u003eUnlike many other extracellular parasites, \\u003cem\\u003eE. histolytica\\u003c/em\\u003e employs a particularly aggressive mechanism of host interaction known as trogocytosis - a process in which the parasite physically nibbles away portions of host cell membranes, leading to gradual cell destruction (Ralston et al., \\u003cspan citationid=\\\"CR79\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). This mechanism differs from phagocytosis as it occurs while the host cells are still alive, preceding their lysis. Additionally, this parasite deploys a diverse array of virulence factors to adapt to the host environment, evade immune defenses, and establish infection (Stanley, \\u003cspan citationid=\\\"CR96\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e). \\u003cem\\u003eE. histolytica\\u003c/em\\u003e secretes cysteine proteases (EhCPs) that degrade host extracellular matrix components, such as collagen, fibronectin, and laminin, facilitating tissue invasion and causing intestinal ulceration (Li et al., \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e1995\\u003c/span\\u003e; Lidell et al., \\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e; Moncada et al., \\u003cspan citationid=\\\"CR68\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Schulte and Scholze, \\u003cspan citationid=\\\"CR87\\\" class=\\\"CitationRef\\\"\\u003e1989\\u003c/span\\u003e). The parasite also releases lectins, such as the Gal/GalNAc lectin, which bind to host glycoproteins and trigger apoptotic and necrotic pathways, further contributing to epithelial damage (Blazquez et al., \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e; Huston et al., \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe pathogenicity of \\u003cem\\u003eE. histolytica\\u003c/em\\u003e is driven by a complex network of virulence factors, many of which remain poorly characterized (Biller et al., \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Faust and Guillen, \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). While cysteine proteases such as EhCP-A5 are well-characterized for their roles in disrupting tight junctions, triggering inflammation, and facilitating tissue invasion, the contributions of other effector proteins remain less understood (El-Dib, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Recently, the KERP family of lysine- and glutamic acid-rich proteins has emerged as a key player in \\u003cem\\u003eE. histolytica\\u0026rsquo;s\\u003c/em\\u003e interaction with host cells. The KERP family includes three proteins, KERP1, 2, and 3, characterized by their high lysine and glutamic acid content. Both KERP1 and KERP2, but not KERP3, have been identified in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e membrane fractions and exhibit the capacity to bind to the brush border of Caco-2 cells, with KERP1 extensively studied as a virulence factor for its role liver abscess formation (Santi-Rocca et al., \\u003cspan citationid=\\\"CR85\\\" class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e; Seigneur et al., \\u003cspan citationid=\\\"CR89\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e)​. While KERP1 was found to only be expressed in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e but not in non-virulent strains such as \\u003cem\\u003eE. dispar\\u003c/em\\u003e, KERP2 is widely present in \\u003cem\\u003eEntamoeba\\u003c/em\\u003e species. Current KERP2 genetic studies suggesting its involvement in amoebic liver abscess and asymptomatic infections through polymorphisms in the \\u003cem\\u003ekerp2\\u003c/em\\u003e locus​ (Das et al., \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). These findings suggest selective evolutionary pressure on KERP2, pointing to its potential role in parasites adaptation, however, the host-interacting functions and mechanisms of KERP2 remains poorly explored.\\u003c/p\\u003e \\u003cp\\u003eOur study suggested that KERP2 acts as a critical regulator balancing the virulence of \\u003cem\\u003eE. histolytica\\u003c/em\\u003e, allowing the host cell cycle to proceed under pressure, and disrupting cytoskeletal dynamics to render the barrier resistance, finally should contribute to the enhancement of parasitic colonization and residence. These insights not only advance our understanding of KERP2\\u0026rsquo;s dual role in \\u003cem\\u003eE. histolytica\\u0026rsquo;s\\u003c/em\\u003e adaptability and pathogenic potential but also highlight its significance as a versatile virulence factor.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIn Silico Analysis of KERP2\\u003c/h2\\u003e \\u003cp\\u003eTo investigate the evolutionary relationships among KERPs, we compared their primary structures and found that KERP2 (EHI_065630) shares 67.5% sequence identity with KERP3 (EHI_198680), whereas both are distinct from KERP1 (EHI_098210). Using KERP2 as a query, BLASTp and HMMER searches identified regional conservation with DEK-like proteins (Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eA). To further examine the evolutionary history of KERP2, we performed maximum likelihood phylogenetic reconstruction using IQ-TREE, incorporating homologous sequences from a broad range of eukaryotic taxa (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). The results revealed that KERP2 and KERP3 are restricted to \\u003cem\\u003eEntamoeba\\u003c/em\\u003e species, forming a well-supported monophyletic group, suggesting lineage-specific conservation. However, their evolutionary relationship to homologs in fungi, algae, plants, and metazoans is poorly supported, indicative of early divergence from other eukaryotic orthologs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). To assess selection pressure acting on KERP2 within \\u003cem\\u003eEntamoeba\\u003c/em\\u003e species, dN/dS (ω) analysis was performed, yielding a ratio of 0.07979, indicating strong purifying selection. This suggests that KERP2 is functionally constrained within \\u003cem\\u003eEntamoeba\\u003c/em\\u003e, maintaining its conserved role rather than undergoing rapid adaptation. Notably, when using KERP2 DNA sequences to search within Amoebozoa, two species, \\u003cem\\u003ePolysphondylium pallidum\\u003c/em\\u003e and \\u003cem\\u003eDictyostelium purpureum\\u003c/em\\u003e, showed partial conservation in specific regions (Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eB). However, these matches were relatively weak, suggesting that KERP2-like sequences may exist as distant remnants in other Amoebozoans but have not been retained as functional orthologs.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eIntracellular localization of KERP2 in\\u003c/b\\u003e \\u003cb\\u003eE. histolytica\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eTo investigate the intracellular localization of KERP2 in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e, we generated an amebic line expressing HA-tagged KERP2 and a truncated variant, HA-KERP2\\u003csup\\u003e∆185\\u0026ndash;239\\u003c/sup\\u003e, which lacks the coiled-coil domain. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). Immunofluorescence analysis (IFA) with line intensity profiling revealed distinct localization patterns (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). Wild-type HA-KERP2 predominantly localized to the nuclear periphery and nucleoplasm, with punctate signals in the cytosol. In contrast, HA-KERP2\\u003csup\\u003e∆185\\u0026ndash;239\\u003c/sup\\u003e exhibited a significant reduction in nuclear localization and was redistributed to the cytosol.\\u003c/p\\u003e \\u003cp\\u003eTo validate these observations, we performed live-cell imaging using GFP-HA-tagged constructs. The results consistent with the IFA data, with GFP-HA-KERP2 predominantly localized in the nucleus (Videos S1), while GFP-HA-KERP2\\u003csup\\u003e∆185\\u0026ndash;239\\u003c/sup\\u003e remained cytosolic (Video S2). Immuno-electron microscopy (EM) further demonstrated HA-KERP2 enrichment in electron-dense nuclear regions, typically associated with chromatin-rich areas (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). Additionally, HA-KERP2 signals were detected in small electron-dense granule (EDG)-like structures, large vesicles, and diffusely distributed in the cytosol (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD).\\u003c/p\\u003e \\u003cp\\u003eTo complement our imaging analyses, we conducted subcellular fractionation to biochemically assess the distribution of KERP2 variants. Cellular fractions were separated into a 14,000 \\u0026times; g pellet (nuclear fraction, containing nuclei, heavy organelles, and membrane components) and a supernatant (cytosolic fraction, containing cytosolic and light vesicular components) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE-F). HA-KERP2 was predominantly enriched in the nuclear fraction, consistent with its nuclear and membrane association. In contrast, HA-KERP2\\u003csup\\u003e∆185\\u0026ndash;239\\u003c/sup\\u003e was primarily detected in the cytosolic fraction, further supporting its cytosolic localization and aligning with our imaging results. Cysteine synthase (CS) and histone were used as cytosolic and nuclear markers, respectively.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eGene silencing of KERP2 reveals its potential role in regulation of parasitic activities\\u003c/h3\\u003e\\n\\u003cp\\u003eTo investigate the functional role of KERP2, we generate a \\u003cem\\u003eKERP2\\u003c/em\\u003e-knockdown strain (psAP-KERP2gs), utilizing small interfering RNAs with the \\u003cem\\u003epsAP-2-Gunma\\u003c/em\\u003e plasmid. Quantitative reverse transcription PCR (qRT-PCR) confirmed a near-complete reduction of \\u003cem\\u003eKERP2\\u003c/em\\u003e transcript levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA, Fig. S2A). Cell growth assays showed no significant differences between the KERP2gs and psAP-mock strains (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB, Fig. S2B-C).\\u003c/p\\u003e \\u003cp\\u003eTo assess transcriptional changes resulting from \\u003cem\\u003eKERP2\\u003c/em\\u003e knockdown, we performed RNA-Seq analysis. Principal component analysis (PCA) showed distinct clustering between KERP2gs and psAP-mock strains (Fig. S2D). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses identified upregulation of genes associated with proteolysis regulation, sulfur amino acid metabolism, and amoebiasis in KERP2gs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC). Genes showing notable increases in expression included two cysteine synthases (EHI_024230, 7.74-fold; EHI_160930, 4.68-fold) and methionine γ-lyase (EHI_057550, 18.3-fold) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD). Additionally, cysteine protease (EHI_010850) was upregulated (2.11-fold), along with three pore-forming peptides (EHI_169350, 4.26-fold; EHI_194540, 2.71-fold; EHI_15940, 2.2-fold).\\u003c/p\\u003e \\u003cp\\u003eTo confirm the regulation role of KERP2 in amoebiasis-related genes, we evaluated changes in cysteine protease (CP) activity. Intracellular CP activity across four \\u003cem\\u003eE. histolytica\\u003c/em\\u003e strains, including HA-KERP2-overexpressing and HA-mock control strains in the G3 background, was measured. KERP2gs strain exhibited a 1.29-fold higher intracellular CP activity (8.05\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.49) compared to psAP-mock (6.25\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.25, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001). In contrast, HA-KERP2 overexpression resulted in a 0.89-fold reduction in intracellular CP activity (5.39\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.19) compared to HA-mock (6.08\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.18, p\\u0026thinsp;=\\u0026thinsp;0.003) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE, Fig. S2E-F). Differences were also observed in released CP activity. The KERP2gs strain exhibited a 6.63-fold increase in released CP activity (7.89\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.15) compared to psAP-mock (1.19\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.12, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001). In contrast, HA-KERP2 overexpression resulted in lower released CP activity, with HA-KERP2 cells showing 0.75-fold the activity of HA-mock cells (1.39\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.35 vs. 1.85\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.47, p\\u0026thinsp;=\\u0026thinsp;0.032) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF, Fig. S2G-H).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eNuclear co-immunoprecipitation revealed KERP2\\u0026rsquo;s role and its trafficking mechanisms in E\\u003c/b\\u003e. \\u003cb\\u003ehistolytica\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eTo elucidate KERP2 function through protein-protein interactions, co-immunoprecipitation (co-IP) followed by mass spectrometry (MS) analysis was performed on four biological replicates. HA-KERP2, HA-KERP2\\u003csup\\u003e∆185\\u0026ndash;239\\u003c/sup\\u003e, and HA-mock samples were fractionated into nuclear and cytosolic fractions before undergoing independent co-IP. Western blotting and Flamingo staining confirmed the success of co-IP (Fig. S3A-D). KERP2 was consistently detected in all HA-KERP2 pull-down samples and identified by MS, except in the nuclear fraction of Trial 3, where non-specific binding in the HA-mock control led to its exclusion from further analysis.\\u003c/p\\u003e \\u003cp\\u003eSignificant KERP2-binding proteins were defined as those exhibiting at least a two-fold increase in quantitative value (QV) in HA-KERP2 or HA-KERP2\\u003csup\\u003e∆185\\u0026ndash;239\\u003c/sup\\u003e compared to HA-mock and appearing in at least two out of four biological replicates. In HA-KERP2 nuclear fraction, 75 proteins were identified as double hits and 4 as triple hits, while its cytosolic fraction contained 14 double-hit and 5 triple-hit proteins (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA-B, Table S3). GO enrichment analysis indicated that nuclear KERP2 interactors were linked to vesicles and non-membrane-bound organelles, including ribosomes, with functional associations to binding, metabolism, and ribosome biogenesis (Fig. S3E-F). Meanwhile, KEGG enrichment analysis of cytosolic interactors highlighted their involvement in ribosome and protein processing in the endoplasmic reticulum (ER) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD).\\u003c/p\\u003e \\u003cp\\u003eTo further characterize the nuclear interactome, proteins were categorized based on functionality beyond KEGG enrichment. They were grouped into clusters related to protein transport, protein folding, nucleic acid binding, and ribosome biogenesis, as visualized in a heat map of their QVs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eE, Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Notably, EhRab11B, EhVPS45A, and clathrin coat assembly protein\\u0026mdash;key players in endosomal trafficking\\u0026mdash;were identified. Proteins associated with nucleic acid binding and ribosome biogenesis were more enriched in HA-KERP2, whereas those involved in protein folding and certain aspects of protein transport were more prominent in HA-KERP2\\u003csup\\u003e∆185\\u0026ndash;239\\u003c/sup\\u003e. The cytosolic interactome also revealed interactions with Granin1, Granin2, and Bip, consistent with nuclear interactors, along with EhC2B (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eF, Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eEnriched and significant nuclear hits in HA-KERP2\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"3\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eDescription\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eFunctionality\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eAccession number\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSjogren's syndrome/scleroderma autoantigen 1 (Autoantigen p27)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_006840\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eEhRab11b\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Transport\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_107250\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGranin 1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Transport\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_167300\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eNucleolar GTP-binding protein 1, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRibosome Biogenesis\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_174940\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCalmodulin, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_000130\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSignal recognition particle protein SRP54, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Transport\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_004750\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eNuclear transport factor 2 domain containing protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Transport/Ribosome Biogenesis\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_035490\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTranscription factor/nuclear export subunit protein 2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eNucleic Acid Binding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_052850\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCell division protein kinase, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_105300\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eEnhancer binding protein-1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eNucleic Acid Binding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_121780\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eHMG box protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eNucleic Acid Binding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_179340\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eRNA recognition motif domain containing protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eNucleic Acid Binding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_026440\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eHistone deacetylase, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eNucleic Acid Binding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_119320\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eChromatin organization modifier domain containing protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eNucleic Acid Binding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_031370\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eEhVPS45A\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Transport\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_160900\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eHSP70 with ER retention signals, predicted as BiP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Folding/ Protein Transport\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_199590\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eHeat shock protein, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Folding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_022620\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eHeat shock protein 90, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Folding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_102270\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eChaperonin containing TCP-1 delta subunit, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Folding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_114120\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e90 kDa heat shock protein, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Folding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_163480\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCysteine protease-C6\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProteolysis\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_127030\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e14-3-3 protein (EhP2)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Transport\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_098280\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eClathrin coat assembly protein, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Transport\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_135430\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGranin 2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Transport\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_167310\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eEukaryotic translation initiation factor 6, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRibosome Biogenesis\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_006170\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eNucleolar phosphoprotein Nopp34, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRibosome Biogenesis\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_068680\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eRibosome biogenesis regulatory protein, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRibosome Biogenesis\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_098810\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGuanine nucleotide-binding protein subunit beta 2-like 1, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_110400\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eArmadillo/beta-catenin-like repeats\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_068510\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eRas family GTPase\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_137700\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eRho family GTPase\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_190440\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eActophorin, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_197480\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eActin, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_198930\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eWD40 repeats\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_103620\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eRab family GTPase\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_143650\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eProfilin, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_176140\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSerine-rich 25 kDa antigen protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_116360\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eProtein tyrosine kinase domain-containing protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_101280\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eMalic enzyme, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_044970\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eLysyl-tRNA synthetase\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_047810\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e2,3-bisphosphoglycerate-independent phosphoglycerate mutase, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_050940\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eL-myo-inositol-1-phosphate synthase\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_070720\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eAlcohol dehydrogenase, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_125950\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ephosphoglycerate kinase, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_188180\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eLipase (class 3), putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_032470\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eV-type ATPase, A subunit, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_043010\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eprotein disulfide isomerase, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_071590\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003efructose-1,6-bisphosphate aldolase, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_098570\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eAlcohol dehydrogenase 3, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_198760\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePyruvate phosphate dikinase\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_009530\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eEnolase, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_130700\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e26S proteasome non-ATPase regulatory subunit, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProteolysis\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_030170\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUbiquitin binding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProteolysis\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_031950\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e26S protease regulatory subunit, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProteolysis\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_080890\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUbiquitin, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProteolysis\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_083270\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e40S ribosomal protein S4\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRibosome\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_118170\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e60S ribosomal protein L13\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRibosome\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_181560\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e60S ribosomal protein L7a, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRibosome\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_029530\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e60S ribosomal protein L18a\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRibosome\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_035600\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e40S ribosomal protein S7, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRibosome\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_067530\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e60S ribosomal protein L11\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRibosome\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_124300\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e60S ribosomal protein L9\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRibosome\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_126140\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eRibosomal protein eS8, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRibosome\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_009870\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e60S ribosomal protein L2/L8, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRibosome\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_127200\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_155400\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_155590\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_017750\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_085010\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_124820\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_155590\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_178780\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_183160\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_189930\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_194870\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_005150\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_053100\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_178970\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_197450\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_122900\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eEnriched and significant cytosolic hits in HA-KERP2\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"3\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eDescription\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eFunctionality\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eAccession number\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e60S ribosomal protein L12, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_030710\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eHeat shock protein 70, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Folding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_052860\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eribosomal protein S25, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_074800\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGrainin 1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Transport\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_167300\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eAcetyl-CoA synthetase, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_178960\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGlyceraldehyde-3-phosphate dehydrogenase, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_008200\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_017690\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e40S ribosomal protein S5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_044590\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eRas family GTPase\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eVesicular Trafficking\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_058090\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eEhC2B\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_059860\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eNucleosome assembly protein, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eNucleic Acid Binding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_072030\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e40S ribosomal protein S15a, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_073600\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eFructose-1,6-bisphosphate aldolase, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_098570\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eActin, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_107290\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eRibosomal protein L11, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_124300\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e40S ribosomal protein S21, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_126870\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eUncharacterized protein\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_146110\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGrainin 2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Transport\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_167310\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e70 kDa heat shock protein, putative\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eProtein Folding\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eEHI_199590\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eGiven the established role of Rab11b in extracellular cysteine protease transport in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e, we performed IFA to confirm its interaction with HA-KERP2. This revealed co-localization of HA-KERP2 with Rab11B-positive vesicles (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eG), supporting its involvement in vesicle-associated trafficking.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eTranslocation of KERP2 from\\u003c/b\\u003e \\u003cb\\u003eE. histolytica\\u003c/b\\u003e \\u003cb\\u003eto host epithelial cells\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eSince KERP1 and KERP2 were first identified as binding to the brush border of Caco-2 cells, we investigated whether KERP2 is truly transferred from \\u003cem\\u003eE. histolytica\\u003c/em\\u003e to host cells and contributes to host-parasite interactions. Co-culturing HA-KERP2-expressing trophozoites with Caco-2 cells for 2 hours, followed by IFA, revealed punctate HA-KERP2 signals in the cytosol and nucleus of Caco-2 cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA). Live imaging of GFP-HA-KERP2-expressing trophozoites moving over the Caco-2 monolayer confirmed GFP signals within Caco-2 cells, appearing in zone 1 and zone 2 between 30 and 45 minutes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB, Video S4, S5). Higher-magnification imaging revealed similar punctate structures (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC). This translocation was absent in control experiments using trophozoites expressing GFP-RtcB2, a cytosolic tRNA ligase (Fig. S4A-B).\\u003c/p\\u003e \\u003cp\\u003eTo validate these observations under more physiological conditions, we used a 3D crypt model with differentiated enterocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD). After 2 hours of interaction, HA-KERP2 signals were detected in enterocytes, whereas GFP-HA remained within trophozoites (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE).\\u003c/p\\u003e \\u003cp\\u003eImmunoblot analysis further confirmed KERP2 translocation. HA-KERP2-expressing trophozoites were co-cultured with a Caco-2 monolayer for 1\\u0026ndash;3 hours, and cells were washed to separate \\u003cem\\u003eE. histolytica\\u003c/em\\u003e from Caco-2 cells (confirmed by CS-1 and GAPDH markers, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eF). Western blotting detected HA-KERP2 in Caco-2 cells after 1 hour, with levels increased at 2 and 3 hours. Notably, galactose treatment, which inhibits \\u003cem\\u003eE. histolytica\\u003c/em\\u003e adhesion, significantly reduced KERP2 translocation.\\u003c/p\\u003e \\u003cp\\u003eTo explore how KERP2 is released, we examined its secretion via extracellular vesicles (EVs). Full-length HA-KERP2 was undetectable in EVs, but HA-KERP2\\u003csup\\u003eΔ185\\u0026ndash;239\\u003c/sup\\u003e and GFP-HA were enriched, which may be due to redundant protein disposal rather than functional secretion (Fig. S4C).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eKERP2 Trafficking Mechanism in\\u003c/b\\u003e \\u003cb\\u003eE. histolytica\\u003c/b\\u003e \\u003cb\\u003eand Caco-2 Cells\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eTo investigate KERP2 trafficking during \\u003cem\\u003eE. histolytica\\u003c/em\\u003e interaction with epithelial cells, we performed immuno-EM to visualize the localization of HA-KERP2 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA). \\u003cem\\u003eE. histolytica\\u003c/em\\u003e expressing HA-KERP2 were co-cultured with Caco-2 for 1 hour. In \\u003cem\\u003eE. histolytica\\u003c/em\\u003e, HA-KERP2 signals were predominantly detected near the contact side with Caco-2 cells, besides the nuclear localization. In Caco-2 cells, HA-KERP2 was primarily observed at the microvillus, within the cytosol, and in endosome-like structures, suggesting active uptake.\\u003c/p\\u003e \\u003cp\\u003eTo further assess the mechanism of KERP2 uptake, we expressed and purified recombinant His-GFP-KERP2 and His-GFP in \\u003cem\\u003eE. coli\\u003c/em\\u003e (Fig. S5A-B). Caco-2 cells were incubated with 3 \\u0026micro;M of either His-GFP-KERP2 or His-GFP in complete EMEM, and GFP uptake was quantified over time using flow cytometry (FACS) after trypsinization (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB-C). Within 1 hour, GFP signals were detected in 4.33% of Caco-2 cells exposed to His-GFP-KERP2, increasing progressively to 25.2% over 24 hours, as also reflected by a rise in median fluorescence intensity (MFI). In contrast, His-GFP alone showed minimal uptake, indicating that KERP2 facilitates selective internalization.\\u003c/p\\u003e \\u003cp\\u003eTo determine whether endocytosis plays a role in KERP2 internalization, we incubated Caco-2 cells with His-GFP-KERP2 or RITC-dextran, a well-established endocytosis marker, at 35.5\\u0026deg;C and 4\\u0026deg;C (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eD). As expected, RITC-dextran uptake was significantly inhibited at the lower temperature, decreasing from 53.7% at 35.5\\u0026deg;C to 1.92% at 4\\u0026deg;C, consistent with the suppression of endocytic activity at reduced temperatures. Similarly, His-GFP-KERP2 internalization was markedly reduced at 4\\u0026deg;C, with GFP signals detected in only 2.25% of cells compared to 16.5% at 35.5\\u0026deg;C, suggesting that KERP2 uptake may occur via micropinocytosis or energy-dependent endocytosis-like mechanism.\\u003c/p\\u003e \\u003cp\\u003eTo assess the persistence of internalized KERP2, we exposed Caco-2 cells to His-GFP-KERP2 or His-GFP for 24 hours, then washed the cells thoroughly with DPBS and replenished the medium (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eE). FACS analysis at 24 and 48 hours post-wash revealed that GFP signals persisted in 11.7% and 7.2% of Caco-2 cells, respectively, suggesting that a fraction of internalized KERP2 remains undegraded or unprocessed within host cells.\\u003c/p\\u003e \\u003cp\\u003eSince KERP2 was not detected in extracellular vesicles (EVs) but was previously identified in membrane fractions, we investigated its potential membrane association. A lipid overlay assay using recombinant His-KERP2 (Fig. S5C-D), but not His-GFP, demonstrated binding affinity for PI(3)P, PI(4)P, PI(5)P, PI(4,5)P\\u003csub\\u003e2\\u003c/sub\\u003e, and PA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eF).\\u003c/p\\u003e\\n\\u003ch3\\u003eFunctional Impact of KERP2 on Host Protein Networks and Gene Expression\\u003c/h3\\u003e\\n\\u003cp\\u003eTo investigate the impact of KERP2 on host epithelial cells, we performed co-IP coupled with MS in three biological replicates to identify KERP2-interacting proteins in Caco-2 cells. Caco-2 cells were co-cultured with HA-KERP2- or HA-mock-expressing \\u003cem\\u003eE. histolytica\\u003c/em\\u003e trophozoites, followed by separation, lysate collection, and co-IP analysis. Significant interactors, defined by a more than 2-fold increase in HA-KERP2 samples in at least two replicates, included 78 double-hit and 5 triple-hit proteins (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA).\\u003c/p\\u003e \\u003cp\\u003eGO enrichment analysis revealed associations with cadherin binding, actin binding, and transcription coactivator binding (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eB). Functional categorization (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eC) highlighted triple-hit interactors PPP6C, PPP6R, and RANGAP1, along with double-hit proteins such as CUL5, UBR5, and MIOS, which are linked to signaling pathways and cell cycle regulation. Several interactors were also involved in cytoskeletal organization and adhesion, including PDLIM1, PFN1, ARPC3, CNN3, LASP1, CLDN4, ITGB1, and CTNNB1. Intracellular trafficking proteins, such as EEA1, SNX5, and DCTN1, were also identified.\\u003c/p\\u003e \\u003cp\\u003eTo assess the effects of KERP2 on host gene expression, we performed RNA-seq on Caco-2 cells co-cultured with HA-KERP2-overexpressing, psAP-KERP2gs, or wild-type \\u003cem\\u003eE. histolytica\\u003c/em\\u003e G3 strains, alongside untreated controls. PCA and volcano plots confirmed distinct clustering among all experimental groups, reflecting condition-specific transcriptional profiles (Fig. S6A-D). To specifically examine KERP2-regulated genes, we prioritized those showing significant differential expression in HA-KERP2- vs. psAP-KERP2gs-treated cells, revealing clear separation in PCA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eD).\\u003c/p\\u003e \\u003cp\\u003eDifferentially expressed genes (adjusted p-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, absolute log2 fold change\\u0026thinsp;\\u0026ge;\\u0026thinsp;1) in HA-KERP2-treated cells included upregulated pro-inflammatory mediators (IL1B, IL36G), signaling adapters (SLA), and heme-related genes (HRG), while cytoskeletal regulators (CYTIP, MYOCD), stress-response factor (NUPR1), and solute transporters (SLC15A3) were downregulated (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eE). Conversely, psAP-KERP2gs-treated cells exhibited increased expression of extracellular matrix components (TNC), contractility regulators (MYL7), and metabolic enzymes (CYP4B1), while cilia-associated (CFAP43), solute transport (SLC2A12), and pentose phosphate pathway (TKTL1) genes were downregulated.\\u003c/p\\u003e\\n\\u003ch3\\u003eEffects of KERP2 on DNA synthetic cycle of the host epithelial cells\\u003c/h3\\u003e\\n\\u003cp\\u003eTo evaluate the effects of KERP2 on host cell cycle progression, EdU incorporation assays were performed in Caco-2 cells following a 2-hour interaction with or without different \\u003cem\\u003eE. histolytica\\u003c/em\\u003e strains: HA-KERP2, HA-mock, KERP2gs, and psAP-mock in the G3 background. After co-culture, \\u003cem\\u003eE. histolytica\\u003c/em\\u003e cells were removed through washes with 2% galactose, and Caco-2 cells were incubated with EdU in complete EMEM for 6 hours. Confocal imaging showed differences in EdU-positive nuclei among experimental groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eA).\\u003c/p\\u003e \\u003cp\\u003eQuantification of EdU-positive nuclei, visualized using Hoechst 33342, showed that HA-KERP2-, HA-mock-, and psAP-mock-exposed cells, each containing exogenous or endogenous KERP2, exhibited higher EdU-positive ratios (48\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.0%, 46\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.3%, and 45\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.3%, respectively) compared with the wild-type Caco-2 control (35\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.2%; p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05). By contrast, Caco-2 cells co-cultured with \\u003cem\\u003eKERP2\\u003c/em\\u003e knockdown (KERP2gs) \\u003cem\\u003eE. histolytica\\u003c/em\\u003e displayed an EdU-positive ratio of 33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.3%, which was not significantly different from the wild-type control (p\\u0026thinsp;=\\u0026thinsp;0.9445). These findings suggest that KERP2 is important for promoting Caco-2 cell proliferation, as silencing of \\u003cem\\u003eKERP2\\u003c/em\\u003e in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e abrogates the effect.\\u003c/p\\u003e\\n\\u003ch3\\u003eEffects of KERP2 on cytoskeleton of the host epithelial cells\\u003c/h3\\u003e\\n\\u003cp\\u003eTo examine the impact of KERP2 on cytoskeletal organization, we stained Caco-2 cells with anti-E-cadherin (tight junctions), phalloidin 594 (F-actin), and Hoechst 33342 (nuclei), after a 2-hour interaction (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eC). Morphological analysis revealed that HA-KERP2-cocultured Caco-2 cells displayed an elongated shape, which was also observed in differentiated enterocytes from the 3D colon-on-chip model (Fig. S7A). Quantification of Caco-2 cell shape using cell form factor and aspect ratio measurements revealed significant changes in HA-KERP2-cocultured cells but not in KERP2gs-cocultured cells compared to controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eD-E). The cell form factor, which measures cellular circularity, was 0.59\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.073 in untreated control cells but was significantly reduced to 0.53\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.091 in HA-KERP2-cocultured cells (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), whereas KERP2gs-cocultured cells exhibited values similar to controls (0.57\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.09). Similarly, aspect ratio measurements revealed a slight but statistically significant increase in HA-KERP2-cocultured cells (1.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.5, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) compared to controls (1.6\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.38), while KERP2gs-cocultured cells remained unchanged (1.6\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.39).\\u003c/p\\u003e \\u003cp\\u003eTo assess potential alterations in Caco-2 cell adhesion, we examined F-actin organization along the basal cell surface (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eF). KERP2gs-cocultured Caco-2 cells exhibited the formation of stress fibers, transitioning from the wild-type resting state. In contrast, HA-KERP2-cocultured cells displayed disrupted stress fiber structures, with thinner and less continuous F-actin along the cell edges, suggesting cytoskeletal remodeling.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEffects of KERP2 on motility of the host epithelial cells\\u003c/h2\\u003e \\u003cp\\u003eTo further investigate the impact of KERP2 on Caco-2 cell motility, we conducted a wound healing assay using 2% FBS to minimize serum-induced proliferation. Caco-2 cells were treated for 2 hours with HA-KERP2, HA-mock, KERP2gs, or psAP-mock strains, washed with 2% galactose, and subsequently monitored for gap recovery over time (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eG). Linear regression analysis of wound closure rates revealed that KERP2gs-treated cells exhibited significantly faster migration (Y = -0.04800*X\\u0026thinsp;+\\u0026thinsp;1.026) compared to psAP-mock-treated cells (Y = -0.04207*X\\u0026thinsp;+\\u0026thinsp;0.9947) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eH). In contrast, HA-KERP2 treatment (Y = -0.03861*X\\u0026thinsp;+\\u0026thinsp;1.048) resulted in a slower migration rate relative to HA-mock treatment (Y = -0.03870*X\\u0026thinsp;+\\u0026thinsp;1.024). Notably, HA-KERP2-treated, HA-mock-treated, and psAP-mock-treated cells, each theoretically expressing different levels of KERP2, migrated more slowly than untreated controls (Y = -0.04339*X\\u0026thinsp;+\\u0026thinsp;1.026), with varying degrees of reduction.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eEffects of KERP2 on the tight junction integrity of the host epithelial cells\\u003c/h3\\u003e\\n\\u003cp\\u003eTo evaluate the impact of KERP2 on epithelial monolayer integrity, transepithelial electrical resistance (TEER) was measured in Caco-2 cells cultured on transwell inserts and exposed to different \\u003cem\\u003eE. histolytica\\u003c/em\\u003e strains (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eA-C, Fig. S7B-E). TEER measurements showed that KERP2gs-cocultured cells exhibited the largest reduction in TEER, with values decreasing by 52.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.5% after 1 hour (from 101.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.7% to 49.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.2%) and by 73.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.5% after 2 hours (to 28.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.2%). In comparison, psAP-mock-cocultured cells exhibited 25.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.5% TEER reduction after 1 hour (from 104.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.5% to 79.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.0%) and 36.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.5% after 2 hours (to 68.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.0%). HA-KERP2-cocultured cells exhibited a 36.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.5% TEER reduction after 1 hour (from 98.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.0% to 62.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.6%) and 65.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.5% after 2 hours (to 33.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.0%). HA-mock-cocultured cells exhibited a 32.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.2% TEER reduction after 1 hour (from 103.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.2% to 71.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.0%) and 54.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.2% after 2 hours (to 49.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.5%).\\u003c/p\\u003e \\u003cp\\u003eTo also assess the impact of recombinant KERP2 on epithelial monolayer integrity, relative TEER (%) was measured at 0, 3, and 24 hours post-treatment with increasing concentrations of His-GFP-KERP2 (1.5 \\u0026micro;M, 3 \\u0026micro;M, 6 \\u0026micro;M) and control His-GFP (3 \\u0026micro;M, 6 \\u0026micro;M) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eD, Fig. S7F-G). TEER values were normalized to baseline before measurement. After 3 hours, His-GFP-KERP2 treatment resulted in a dose-dependent TEER reduction. The 1.5 \\u0026micro;M treatment led to a 14.9% decrease (from 117.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;7.4% to 100.1\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;6.2%), while the 3 \\u0026micro;M treatment caused an 18.3% reduction (from 107.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;7.1% to 87.8\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;6.6%). The most significant decrease was observed with 6 \\u0026micro;M His-GFP-KERP2, where TEER dropped by 77.7% (from 113.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.0% to 36.2\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.3%). His-GFP controls exhibited only minor reductions, with decreases of 4.6% (3 \\u0026micro;M) and 9.7% (6 \\u0026micro;M). At 24 hours, TEER reductions were more pronounced across all His-GFP-KERP2 treatments. The 1.5 \\u0026micro;M concentration led to a 43.0% decrease (from 117.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;7.4% to 74.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.8%), while the 3 \\u0026micro;M and 6 \\u0026micro;M concentrations resulted in reductions of 44.9% and 80.3%, respectively. Control groups showed smaller reductions, with TEER decreasing by 30.1% (3 \\u0026micro;M His-GFP) and 35.1% (6 \\u0026micro;M His-GFP).\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003e \\u003cem\\u003eE. histolytica\\u003c/em\\u003e heavily relies on adhesion to epithelial cells to initiate colonization and infection, a process mediated by interactions between trophozoite plasma membrane and host brush border microvilli (Seigneur et al., \\u003cspan citationid=\\\"CR89\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e). KERP2, identified alongside KERP1, was predicted to localize in the extracellular milieu, despite lacking a transmembrane domain or GPI anchor. Its high isoelectric point (pI = 9.75) and positively charged surface suggest interactions with negatively charged molecules, potentially facilitating adhesion or binding at the host-pathogen interface. Phylogenetic analysis shows KERP2 contains a conserved SAP domain, similar to DEK protein associated with chromatin remodeling and transcription regulation (Aravind and Koonin, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2000\\u003c/span\\u003e; Sanden and Gullberg, \\u003cspan citationid=\\\"CR83\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). DEK proteins, conserved among multicellular eukaryotes and mammals, contrast with their sparse presence in protozoa, where only \\u003cem\\u003eTrypanosoma brucei\\u003c/em\\u003e (TbSAP) exhibits similar SAP domain involved in gene repression (Davies et al., \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e; Smith et al., \\u003cspan citationid=\\\"CR94\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). KERP2 demonstrates functional divergence within Amoebozoa, with lineage-specific evolution indicated by homolog searches across multiple kingdoms revealing no close relatives outside \\u003cem\\u003eEntamoeba\\u003c/em\\u003e. This uniqueness is underscored by strong purifying selection shown through dN/dS analysis, suggesting conserved function with minimal adaptive changes. Searches within Amoebozoa reveal only weakly conserved KERP2-like sequences in \\u003cem\\u003eP. pallidum\\u003c/em\\u003e and \\u003cem\\u003eD. purpureum\\u003c/em\\u003e, indicating that while KERP2-like sequences exist, they do not function as orthologs, emphasizing a specialized role and distinct phylogenetic status of KERP2 within \\u003cem\\u003eEntamoeba\\u003c/em\\u003e.\\u003c/p\\u003e \\u003cp\\u003eThe localization of KERP2 in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e was confirmed using multiple approaches, including IFA, live-cell imaging, immuno-EM, and co-IP. IFA with line intensity profiling demonstrated that HA-KERP2 predominantly localizes at the nuclear periphery and within the nucleoplasm, with punctate signals observed in the cytosol. In contrast, the truncated HA-KERP2\\u003csup\\u003e∆185–239\\u003c/sup\\u003e variant, lacking the coiled-coil domain, was mainly detected in the cytosol, indicating that the C-terminal region is crucial for nuclear localization. Live-cell imaging using GFP-HA-tagged constructs further supported these findings, revealing strong nuclear enrichment of full-length KERP2 but cytosolic redistribution of the truncated variant. Immuno-EM confirmed the presence of HA-KERP2 in electron-dense nuclear regions, which are typically associated with chromatin-rich areas, as observed in other eukaryotic systems where electron-dense regions correspond to heterochromatin at the nuclear periphery or near nucleoli (Ralph et al., \\u003cspan citationid=\\\"CR78\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e; Zuleger et al., \\u003cspan citationid=\\\"CR117\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). Nuclear co-IP further corroborated the nuclear presence of KERP2 by identifying key nuclear-associated binding partners, including transcription-related proteins such as enhancer binding protein-1, HMG box protein, and histone deacetylase, as well as nuclear transport proteins such as SRP54, nuclear transport factor 2 domain-containing protein, and nucleolar GTP-binding protein 1.\\u003c/p\\u003e \\u003cp\\u003eThe nuclear localization of KERP2 suggests a potential role in transcriptional regulation, particularly in chromatin organization. Given that the \\u003cem\\u003eE. histolytica\\u003c/em\\u003e nuclear periphery may contain nucleolus (Jhingan et al., \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e), KERP2 could participate in ribosome biogenesis. However, the lack of a clear nucleolar marker in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e and the transcriptional changes observed upon \\u003cem\\u003eKERP2\\u003c/em\\u003e knockdown, as revealed by RNA-seq, led us to focus more on its potential DNA-binding activity instead. Structurally, KERP2 shares features with DEK proteins, which associate with DNA in a structure-dependent manner rather than through sequence-specific binding (Bohm et al., \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e; Waldmann et al., \\u003cspan citationid=\\\"CR102\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Waldmann et al., \\u003cspan citationid=\\\"CR103\\\" class=\\\"CitationRef\\\"\\u003e2002\\u003c/span\\u003e). They preferentially associate with highly expressed, ubiquitous genes without binding to specific DNA motifs (Sanden et al., \\u003cspan citationid=\\\"CR84\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e), a feature that may extend to KERP2. This hypothesis is supported by chromatin immunoprecipitation (ChIP) assays, where GFP-HA-KERP2 successfully pulled down detectable amounts of DNA, whereas GFP-HA-mock controls did not. However, no specific DNA motifs were identified, with the highest observed enrichment reaching only 3.89-fold compared to input controls (Table S6). Furthermore, all enriched regions were located within coding sequences and lacked distinct motif features. These findings suggest that, similar to DEK proteins, KERP2 may primarily associate with structured chromatin regions rather than specific DNA sequences.\\u003c/p\\u003e \\u003cp\\u003eThis chromatin association is consistent with RNA-seq results, which showed significant transcriptional changes upon \\u003cem\\u003eKERP2\\u003c/em\\u003e knockdown, particularly in genes associated with proteolysis regulation, sulfur amino acid metabolism, and amoebiasis. Notably, cysteine synthases (EHI_024230, EHI_160930) and methionine γ-lyase (EHI_057550) were highly upregulated, suggesting a shift in sulfur metabolism, which is critical for redox balance and oxidative stress resistence in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e during host interaction (Jeelani et al., \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Tokoro et al., \\u003cspan citationid=\\\"CR101\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e). Concomitantly, virulence-associated factors such as cysteine protease (EHI_010850) and pore-forming peptides (EHI_169350, EHI_194540, EHI_15940) were also upregulated. Notably, EHI_010850 encodes an amino acid sequence with 99.7% identity to EhCP-A7 (EHI_039610). Cysteine protease activity assays further supported this regulatory role: KERP2 knockdown significantly increased both intracellular and secreted cysteine protease activity, while KERP2 overexpression resulted in a slight reduction.\\u003c/p\\u003e \\u003cp\\u003eThese findings suggest that KERP2 may function as a chromatin organizer that modulates virulence factor expression. Cysteine proteases and amoebapores are well-established virulence factors in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e, promoting host cell degradation, tissue invasion, and immune evasion (Begum et al., \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Leippe, \\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Moncada et al., \\u003cspan citationid=\\\"CR68\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Singh et al., \\u003cspan citationid=\\\"CR92\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). The upregulation of these virulence factors in \\u003cem\\u003eKERP2\\u003c/em\\u003e knockdown cells suggests that KERP2 may act as a negative regulator of virulence gene expression. In axenic \\u003cem\\u003eE. histolytica\\u003c/em\\u003e cultures, CP-A1, CP-A2, CP-A5, and CP-A7 account for over 90% of proteolytic activity in trophozoite extracts (Irmer et al., \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). Particularly, EHI_010850 and EhCP-A7 are both significantly upregulated upon contact with human colon explants (Thibeaux et al., \\u003cspan citationid=\\\"CR100\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e), hinting that KERP2-mediated repression of virulence factors could be relevant to the repression of direct host damage.\\u003c/p\\u003e \\u003cp\\u003eA key question is whether the nuclear-cytoplasmic trafficking of KERP2 (further discussed below) serves as a regulatory mechanism that balances virulence factor expression and metabolic adaptation. We hypothesize that during host cell contact, a portion of KERP2 is translocated to host cells, leading to a reduction of its nuclear ratio in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e. This reduction in nuclear KERP2 may relieve transcriptional repression, thereby upregulating virulence genes while concurrently enhancing sulfur metabolism to support increased redox homeostasis. This dual response–activation of cysteine proteases for extracellular matrix degradation and metabolic adaptation for stress resistance–could enable \\u003cem\\u003eE. histolytica\\u003c/em\\u003e to fine-tune its pathogenic potential in response to environmental cues. Future research should investigate whether nuclear KERP2 levels regulate these processes in a dose-dependent manner and whether its redistribution upon host interaction acts as a dynamic switch controlling both virulence and metabolic adaptation.\\u003c/p\\u003e \\u003cp\\u003eKERP2 exhibits a dynamic intracellular distribution in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e, with immuno-EM localization revealing its presence in the nucleus, vesicular structures, and electron-dense granule (EDG)-like compartments within the cytosol. Despite being localized in cytosolic vesicles, full-length KERP2 was not detected in the EV fraction under resting conditions. co-IP analysis identified multiple trafficking-related interactors, including EhRab11B, signal recognition particle protein SRP54, EhVPS45, and clathrin coat assembly protein, suggesting that KERP2 may be transported via recycling endosomes or endosomal sorting pathways. Recycling endosomes, regulated by Rab11B, play a key role in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e vesicular trafficking and secretion of virulence factors such as cysteine proteases (Mitra et al., \\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e). The presence of KERP2 in Rab11B-positive vesicles, but its absence in crude EV fractions, suggests that it may follow an unconventional secretion pathway, likely mediated by recycling endosome-dependent exocytosis rather than exosome release.\\u003c/p\\u003e \\u003cp\\u003eOne possible explanation for the selective secretion of KERP2 is its preferential association with the plasma membrane, potentially due to its positively charged surface (Goldenberg and Steinberg, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e; Woolfson, \\u003cspan citationid=\\\"CR109\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Lipid overlay assays demonstrated that recombinant His-GFP-KERP2 strongly binds to phosphoinositides, including PI3P, PI4P, and PI(4,5)P2, which are enriched in endosomal and plasma membranes (Posor et al., \\u003cspan citationid=\\\"CR77\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). This suggests that KERP2 may be retained at lipid microdomains involved in host-parasite interactions. This hypothesis is further supported by the Immuno-EM, showing KERP2 localization at the plasma membrane, particularly at the parasite-host interface, reinforcing the role of KERP2 as a surface-associated effector rather than being freely secreted.\\u003c/p\\u003e \\u003cp\\u003eTo answer a critical question whether KERP2 can be translocated into host cells during \\u003cem\\u003eE. histolytica\\u003c/em\\u003e-epithelial interactions, IFA, live-cell imaging, and immunoblot analysis of \\u003cem\\u003eE. histolytica\\u003c/em\\u003e-Caco-2 co-cultures were performed. HA-KERP2 and GFP-HA-KERP2 signals were detected inside Caco-2 cells and in a 3D enterocyte crypt model, appearing as punctate cytosolic structures, confirming its uptake. Notably, inhibiting \\u003cem\\u003eE. histolytica\\u003c/em\\u003e adhesion abolished KERP2 translocation, reinforcing the contact-dependent nature of its release from \\u003cem\\u003eE. histolytica\\u003c/em\\u003e. Immuno-EM showed HA-KERP2 within microvilli, the cytosol, and endosome-like vesicles, reinforcing that KERP2 enters the host through an active endocytic process. This was further validated by recombinant His-GFP-KERP2 uptake assays, where purified His-GFP-KERP2 induced a fluorescent increase in Caco-2 cells, quantified by FACS. Trypsinization before FACS ensured that KERP2 was truly internalized rather than merely adhering to the cell surface. Notably, uptake was significantly reduced at 4°C, indicating that KERP2 entry is energy-dependent and mediated by endocytosis.\\u003c/p\\u003e \\u003cp\\u003eThe persistence of KERP2 within punctate cytosolic structures raises the question of whether it remains in endosomal compartments or escapes into the cytosol. The retention of recombinant His-GFP-KERP2 in Caco-2 cells after 48 hours suggests that it is not simply targeted for lysosomal degradation. Instead, its interaction with host endocytic regulators (EEA1, SNX5), as revealed by HA-KERP2 co-IP in Caco-2 cells, suggests potential crosstalk with host vesicular trafficking pathways. Additionally, HA-KERP2 co-IP in Caco-2 cells identified potential receptors for endocytosis, including claudin-4 (CLDN4) and integrin beta-1 (ITGB1), both of which have been demonstrated to be exploited by pathogens for host cell internalization. CLDN4 serves as a receptor for \\u003cem\\u003eClostridium perfringens\\u003c/em\\u003e enterotoxin (CPE), enabling toxin binding and tight junction disruption (Sonoda et al., \\u003cspan citationid=\\\"CR95\\\" class=\\\"CitationRef\\\"\\u003e1999\\u003c/span\\u003e), while ITGB1 has been hijacked by \\u003cem\\u003eStreptococcus pneumoniae\\u003c/em\\u003e and human papillomavirus (HPV) to facilitate adhesion and invasion (De Gaetano et al., \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Woodham et al., \\u003cspan citationid=\\\"CR108\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). Given that KERP2 is internalized via an endocytic pathway, these receptors may mediate its uptake, however, further validation is required.\\u003c/p\\u003e \\u003cp\\u003eOur observation that recombinant His-GFP-KERP2 remained undegraded in Caco-2 cells for up to 48 hours suggests that KERP2 may have specific, active functions within host epithelial cells. To explore these potential roles, we performed co-IP assays, which identified a range of host proteins interacting with KERP2. These include regulators of the cell cycle and cytoskeletal organization. Complementary RNA-seq analyses showed differential expression of genes linked to stress responses and cytoskeletal remodeling in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e-exposed Caco-2 cells. In details, cells treated with HA-KERP2 exhibited upregulation of pro-inflammatory mediators and signaling adapters, alongside downregulation of cytoskeletal regulators and stress-response genes. In contrast, psAP-KERP2gs-treated cells showed elevated expression of extracellular matrix components and contractility regulators. These data point to two major pathways, cell cycle regulation and cytoskeletal dynamics, being prominently affected by KERP2. Specifically, the interaction of KERP2 with PPP6C, the catalytic subunit of protein phosphatase 6 (PP6), may underlie changes in cell cycle progression. PP6 is known to regulate DNA damage repair and inflammatory signaling (Ohama, \\u003cspan citationid=\\\"CR73\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e), while its regulatory subunits (PPP6R) stabilize PPP6C and modulate localization and substrate specificity (Ohama et al., \\u003cspan citationid=\\\"CR74\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e). Notably, PP6 levels increase with cell density in epithelial cells, where PP6 may interact with the E-cadherin cytoplasmic tail to support cell-cell adhesion (Ohama et al., \\u003cspan citationid=\\\"CR74\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e). However, the precise triggers for PP6 upregulation and its functional outcomes remain unresolved, and PPP6C deficiency appears to have opposing effects across different cell types. In mouse embryonic fibroblasts, PPP6C depletion causes G1/S arrest (Ohama et al., \\u003cspan citationid=\\\"CR74\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e), whereas in primary keratinocytes, PPP6C loss promotes S-phase entry and proliferation (Hayashi et al., \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Yan et al., \\u003cspan citationid=\\\"CR111\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eIn parallel, the interactions of KERP2 with cytoskeletal regulators provide another line of evidence for functional perturbations in actin dynamics. PFN1 (profilin 1) and ARPC3 (a subunit of the ARP2/3 complex) both have direct, well-documented roles in actin polymerization (Goley and Welch, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e; Pizarro-Cerda et al., \\u003cspan citationid=\\\"CR76\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Rotty et al., \\u003cspan citationid=\\\"CR82\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e; Witke, \\u003cspan citationid=\\\"CR107\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). PFN1 recharges actin monomers by promoting the exchange of ADP for ATP, while ARPC3 mediates new actin filament nucleation and branching. Additionally, LASP1 and PDLIM1, although not direct drivers of actin polymerization, act as scaffolding proteins influencing cellular migration, focal adhesion, and overall cytoskeletal organization (Orth et al., \\u003cspan citationid=\\\"CR75\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Zhou et al., \\u003cspan citationid=\\\"CR115\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eFrom these findings, we hypothesize that the perturbation of key molecules for signaling pathways that could affect cell cycle and cytoskeletal regulation may be primary consequences of KERP2 in Caco-2 cells. Nevertheless, it is important to note that other signaling axes, such as NF-κB, mTOR, and multiple ubiquitin-mediated pathways, also emerged from the co-IP data and are likely to integrate with or modulate the effects on cell cycle and cytoskeleton. Further experimental validation of these pathways will be necessary to elucidate the full range of KERP2 influence on host cell physiology.\\u003c/p\\u003e \\u003cp\\u003eThe observed increase in EdU-positive nuclei in KERP2-expressing \\u003cem\\u003eE. histolytica\\u003c/em\\u003e-cocultured Caco-2 cells suggests that KERP2 modulates host cell cycle progression. While knockdown of \\u003cem\\u003eKERP2\\u003c/em\\u003e (KERP2gs) had no detectable effect on the G1/S transition, the presence of KERP2 led to a greater proportion of cells entering S phase. One possibility is that KERP2 inhibits PP6 activity, thereby promoting proliferation; however, the precise mechanism by which PP6 regulates the cell cycle remains unclear. Alternatively, enhanced proliferation could be a downstream effect of altered signaling or disrupted cytoskeletal architecture. Consistent with these notions, RNA-seq data from HA-KERP2-treated cells showed upregulation of inflammatory mediators, signaling adapters, and downregulation of cytoskeletal regulators, stress-response genes, and solute transporters, as discussed above. Such transcriptional changes may create an environment more conducive to S-phase entry. For instance, diminished expression of CYTIP and MYOCD could relax the cytoskeletal constraints that normally govern cell cycle checkpoints (Ingber, \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e1993\\u003c/span\\u003e; Mammoto and Ingber, \\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e), while elevated inflammatory signals (IL1B, IL36G) might activate proliferative or survival pathways (Karin and Greten, \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e). Nevertheless, these findings imply that KERP2 provides \\u003cem\\u003eE. histolytica\\u003c/em\\u003e with the capacity to promote the G1/S transition in host epithelial cells, and this effect is significantly reduced when \\u003cem\\u003eKERP2\\u003c/em\\u003e is knocked down.\\u003c/p\\u003e \\u003cp\\u003eFunctional assays demonstrate that KERP2 disrupts host cytoskeletal dynamics. Specifically, Caco-2 cells co-cultured with HA-KERP2 exhibit an elongated shape and disrupted stress fibers, suggesting that KERP2 directly manipulates actin networks and junctional complexes. Moreover, recombinant His-GFP-KERP2 alone was sufficient to reduce TEER, indicating that cytoskeletal disruption by KERP2 does not necessarily require high levels of cysteine protease activity. In contrast, KERP2gs strain likely trigger a protease-driven route to epithelial damage. These parasites secrete elevated levels of cysteine proteases as discussed above, which degrade tight junction proteins and extracellular matrix components, leading to a pronounced TEER reduction. However, the absence of KERP2 also appears to allow host cells to mount a compensatory cytoskeletal response, as evidenced by robust stress fiber formation and faster wound closure. These findings suggest that KERP2gs-induced damage elicits a protective or reparative actin remodeling response.\\u003c/p\\u003e \\u003cp\\u003eThus, we hypothesized that \\u003cem\\u003eE. histolytica\\u003c/em\\u003e employs two distinct strategies to compromise epithelial integrity: (i) direct actin disruption by KERP2 and (ii) protease-mediated tight junction. While HA-KERP2 strain secretes less proteases, they still compromise barrier function through cytoskeletal alterations. Conversely, \\u003cem\\u003eKERP2\\u003c/em\\u003e-silenced parasites cause significant junctional damage via proteases but inadvertently trigger stronger host actin-mediated repair mechanisms. These observations highlight KERP2 as a key effector that modulates the mode of epithelial barrier disruption. Future studies should investigate the regulatory circuits controlling KERP2 expression and protease secretion and assess whether cytoskeletal remodeling enhances parasite invasiveness or reflects an adaptive host countermeasure.\\u003c/p\\u003e \\u003cp\\u003eOverall, our study identifies KERP2 as a dual-function effector that regulates both \\u003cem\\u003eE. histolytica\\u003c/em\\u003e homeostasis and host epithelial responses, shedding light on its multifaceted role in parasitism and virulence. By simultaneously disrupting actin networks and junctional complexes while modulating cysteine proteases levels, KERP2 fine-tunes a balance between cytoskeletal perturbation and protease-mediated degradation to evade host defenses. These findings reveal how \\u003cem\\u003eE. histolytica\\u003c/em\\u003e employs both direct cytoskeletal manipulation and protease-driven mechanisms to compromise epithelial barrier integrity and establish infection. Beyond amoebiasis, this study provides a broader framework for understanding how extracellular pathogens utilize multifunctional effectors to regulate virulence and manipulate host responses. The discovery of KERP2’s dual roles highlight potential parallels in other pathogens that fine-tune their pathogenicity by integrating host manipulation with self-transcriptional control. Future research into the regulatory mechanisms governing KERP2 function could yield valuable insights into host-pathogen interactions and inform the development of targeted therapeutic strategies.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section3\\\"\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e \\u003cdiv id=\\\"Sec23\\\" class=\\\"Section3\\\"\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec24\\\" class=\\\"Section2\\\"\\u003e \\u003cdiv id=\\\"Sec25\\\" class=\\\"Section3\\\"\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec26\\\" class=\\\"Section3\\\"\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec27\\\" class=\\\"Section3\\\"\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\n\\n \\u003cdiv id=\\\"Sec32\\\" class=\\\"Section2\\\"\\u003e \\u003cdiv id=\\\"Sec33\\\" class=\\\"Section3\\\"\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec34\\\" class=\\\"Section3\\\"\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\\n\\n\\n\\n\"},{\"header\":\"Methods\",\"content\":\"\\u003ch2\\u003eStructural Modeling, Domain Identification, and Biochemical Property Analysis\\u003c/h2\\u003e\\u003cp\\u003eThe three-dimensional structure of KERP2 was modeled using I-TASSER (Yang and Zhang, \\u003cspan citationid=\\\"CR113\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Zheng et al., \\u003cspan citationid=\\\"CR114\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e; Zhou et al., \\u003cspan citationid=\\\"CR116\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Structural visualization and refinement were performed using PyMOL (The PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC.). Secondary structural motifs, including a coiled-coil domain spanning amino acids 178–216, were predicted using Marcoil and PCOILS (Delorenzi and Speed, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2002\\u003c/span\\u003e; Gabler et al., \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Lupas, \\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e1996\\u003c/span\\u003e; Lupas et al., \\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e1991\\u003c/span\\u003e). The SAP domain of KERP2 was manually aligned based on residue conservation patterns described in (Aravind and Koonin, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2000\\u003c/span\\u003e). Automated alignment attempts using tools such as CLC Viewer (QIAGEN Bioinformatics, Aarhus, Denmark) and PSI-BLAST did not yield satisfactory results due to the weak sequence conservation of the SAP domain. To address this, residues in KERP2 were manually examined and aligned to known SAP domains following the consensus criteria outlined in the reference study. Key features used for the alignment included: hydrophobic or aliphatic residues (YFWLIVMA), small residues (SAGTVPNHD), polar residues (STQNEDRKH), and bulky residues (KREQWFYLMI). The alignment process was further varified by predicted secondary structure features by PHD program (Rost and Sander, \\u003cspan citationid=\\\"CR81\\\" class=\\\"CitationRef\\\"\\u003e1994\\u003c/span\\u003e). Nuclear localization signals (NLS) were mapped using the NLS Mapper tool (Kosugi et al., \\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). The isoelectric point (pI) of KERP2 was calculated using ExPASy’s Compute pI/Mw tool (Gasteiger et al., \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e). Surface charge distribution was analyzed by combining the predicted structure from I-TASSER with visualization and electrostatics tools in VMD and APBS (Humphrey et al., \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e1996\\u003c/span\\u003e; Jurrus et al., \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e).\\u003c/p\\u003e\\u003ch2\\u003ePhylogenetic Analysis\\u003c/h2\\u003e\\u003cp\\u003eBLASTp and HMMER searches were performed to identify KERP2 orthologs across a broad range of taxa using KERP2 as a query. The obtained sequences were aligned using MAFFT v7.475 (Katoh and Standley, \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e) and trimmed by TrimAl v1.4 (Capella-Gutierrez et al., \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). Phylogenetic reconstruction was conducted using the maximum likelihood method in IQ-TREE v2.3.6 (Minh et al., \\u003cspan citationid=\\\"CR66\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e) with default parameters. The resulting phylogenetic tree was visualized using iTOL v7.0 (Letunic and Bork, \\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). The ratio of nonsynonymous (dN) to synonymous (dS) substitutions (ω = dN/dS) was calculated using the codeml module in PAML v4.9.\\u003c/p\\u003e\\u003ch2\\u003ePlasmid Construction\\u003c/h2\\u003e\\u003cp\\u003eTotal RNA from \\u003cem\\u003eE. histolytica\\u003c/em\\u003e trophozoites was extracted using the TRIzol reagent (Invitrogen) following the manufacturer’s protocol. Briefly, cells were lysed directly in TRIzol reagent, and the homogenized lysate was incubated at room temperature for 5 minutes to allow complete dissociation of nucleoprotein complexes. Chloroform (0.2 mL per 1 mL of TRIzol) was added to the lysate, vortexed vigorously for 15 seconds, and incubated at room temperature for 3 minutes. The mixture was centrifuged at 12,000 × g for 15 minutes at 4°C to separate phases. The aqueous phase containing RNA was transferred to a new tube, and RNA was precipitated by adding isopropanol (0.5 mL per 1 mL of TRIzol), incubated at room temperature for 10 minutes, and centrifuged at 12,000 × g for 10 minutes at 4°C. The RNA pellet was washed with 75% ethanol (1 mL per 1 mL of TRIzol) and centrifuged at 7,500 × g for 5 minutes at 4°C. After air-drying, the pellet was dissolved in RNase-free water and quantified using a NanoDrop spectrophotometer (ThermoFisher). Messenger RNA (mRNA) was purified, and cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System (Invitrogen, ThermoFisher) according to the manufacturer’s protocol.\\u003c/p\\u003e\\u003cp\\u003eThe protein-coding region of \\u003cem\\u003eKERP2\\u003c/em\\u003e was amplified by PCR from \\u003cem\\u003eE. histolytica\\u003c/em\\u003e cDNA using specific oligonucleotides summarized in Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e. To express KERP2 fused with an HA tag at the amino terminus in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e trophozoites, PCR fragments were digested with XmaI and XhoI, purified, and ligated into XmaI- and XhoI-digested pEhExHA vector (Nakada-Tsukui et al., \\u003cspan citationid=\\\"CR71\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e) using cohesive ends, producing the plasmid pEhEx-HA-KERP2.\\u003c/p\\u003e\\u003cp\\u003eTo generate deletion and fusion constructs:\\u003c/p\\u003e\\u003cp\\u003epEhEx-HA-KERP2\\u003csup\\u003e∆185–239\\u003c/sup\\u003e: Reverse PCR was performed on pEhEx-HA-KERP2 using paired primers, followed by blunt-end ligation with T4 Polynucleotide Kinase and T4 DNA Ligase.\\u003c/p\\u003e\\u003cp\\u003epEhEx-GFP-HA-KERP2: GFP was amplified from the pEhEx-GFP vector (Nakada-Tsukui et al., \\u003cspan citationid=\\\"CR71\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e) with BglII end, then ligated into pEhEx-HA-KERP2 digested with BglII.\\u003c/p\\u003e\\u003cp\\u003epEhEx-GFP-HA-KERP2\\u003csup\\u003e∆185–239\\u003c/sup\\u003e: GFP was amplified from pEhEx-GFP and inserted into pEhEx-HA-KERP2\\u003csup\\u003e∆185–239\\u003c/sup\\u003e same as described above.\\u003c/p\\u003e\\u003cp\\u003eTo silence \\u003cem\\u003eKERP2\\u003c/em\\u003e in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e using antisense small RNA, a 420 base-pair fragment corresponding to the 5’ sequence of \\u003cem\\u003eKERP2\\u003c/em\\u003e was amplified by PCR from cDNA using appropriate primers. The amplified fragment was digested with StuI and SacI and cloned into StuI/SacI-digested psAP2-Gunma vector (Mi-Ichi et al., 2011), generating the plasmid psAP2-KERP2gs.\\u003c/p\\u003e\\u003cp\\u003eTo express recombinant KERP2, GFP-KERP2 and GFP with a histidine tag at the amino terminus in bacteria, the KERP2 coding region was first inserted into pEhEx-GFP using appropriate primers with XmaI and XhoI, following the same procedure as described for the construction of pEhEx-HA-KERP2. PCR was then performed on the resulting pEhEx-GFP-KERP2 construct to amplify the GFP-KERP2 fragment, which was subsequently inserted into the pET-151 vector using the In-Fusion HD Cloning Kit (Clontech Laboratories, CA, USA), generating pET-151-GFP-KERP2. A 5× GA linker with XmaI ends, synthesized by FASMAC Co., Ltd. (Kanagawa, Japan), was inserted between GFP and KERP2. Finally, reverse PCR on pET-151-GFP-KERP2 using specific primers was performed to construct pET-151-GFP.\\u003c/p\\u003e\\u003cp\\u003eAll constructs were validated by Sanger sequencing to ensure accurate cloning.\\u003c/p\\u003e\\u003ch2\\u003eCell Culture and Transfection\\u003c/h2\\u003e\\u003cp\\u003eTrophozoites of the \\u003cem\\u003eE. histolytica\\u003c/em\\u003e strain HM-1:IMSS cl-6 (Diamond et al., \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e1978\\u003c/span\\u003e) and G3 (Bracha et al., \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e) were maintained axenically in Diamond’s BI-S-33 medium (Diamond et al., \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e1978\\u003c/span\\u003e) at 35.5°C. Plasmids encoding HA-tagged and GFP-HA-fused KERP2 or its variants, generated as described above, were introduced into HM-1 or G3 trophozoites via lipofection, following the protocol established by (Nozaki et al., \\u003cspan citationid=\\\"CR72\\\" class=\\\"CitationRef\\\"\\u003e1999\\u003c/span\\u003e). Transfected trophozoites were selected and maintained in medium containing 10 µg/mL of G418 (#11811031, Gibco/Life Technologies, Waltham, MA, USA). Plasmids for gene silencing experiments were introduced into the G3 strain by the same lipofection method, and the transfectants were also maintained in medium supplemented with 10 µg/mL of G418. Protein expression was verified by western blotting as described below.\\u003c/p\\u003e\\u003cp\\u003eCaco-2 cells (ATCC) were cultured in Eagle's Minimum Essential Medium (EMEM; ATCC 30-2003) supplemented with 20% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA.) as complete EMEM. The medium was replaced every two days, and cells were incubated under standard cell culture conditions at 37°C in a humidified atmosphere with 5% CO₂.\\u003c/p\\u003e\\u003ch2\\u003eEstablishment of 3D Crypt Model\\u003c/h2\\u003e\\u003cp\\u003eThe 3D crypt model was established by following (Hinman et al., \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Primary intestinal crypts were first isolated and cultivated in collagen-based scaffolds. Crypts were initially dissociated from intestinal tissue using a crypt isolation buffer containing EDTA and dithiothreitol (DTT). The suspension was centrifuged at 600 g for 1 minute, and the pellet was resuspended in pre-warmed maintenance medium (MM) at a concentration of 240 crypts/mL. Crypts were seeded into 6-well plates pre-coated with neutralized collagen hydrogels, which were prepared by combining acid-solubilized collagen with a neutralization buffer (pH 7.4) and allowing the hydrogels to set for 1 hour at 37°C in a humidified CO₂ incubator. The seeded crypts were cultured in MM at 37°C with medium changes every 48 hours until confluency (~ 80%) was achieved. Upon confluency, the cells were passaged by mechanically fragmenting the crypt clusters using a double pipette tip technique and reseeded into fresh collagen-coated scaffolds. To construct the 3D crypt arrays, micromolded collagen scaffolds were fabricated using PDMS molds to mimic the architectural features of in vivo intestinal crypts. These scaffolds were crosslinked and coated following established protocols to ensure mechanical stability and optimal cell attachment. Crypts were seeded into these scaffolds in differentiation medium supplemented with 10 µM Y-27632 to enhance cell survival and attachment. Cultures were maintained in a humidified CO₂ incubator at 37°C, with media changes every 48 hours. Differentiation was induced by establishing a growth-factor gradient across the 3D scaffolds. WRN medium was supplied to the basal reservoir, and differentiation medium was added to the luminal reservoir to promote compartmentalization of stem/proliferative and differentiated cells. The gradient was maintained for 4 days, enabling the formation of a spatially organized crypt-villus axis. After differentiation, the 3D crypts were prepared for imaging and functional analyses. The scaffolds were compatible with confocal microscopy and other phenotypic assays, ensuring their utility for downstream applications.\\u003c/p\\u003e\\u003ch2\\u003eImmunofluorescence Assays (IFA)\\u003c/h2\\u003e\\u003cp\\u003eImmunofluorescence assays were performed to analyze the localization of KERP2 and its variants in \\u003cem\\u003eE. histolytica\\u003c/em\\u003e or mammalian cells.\\u003c/p\\u003e\\u003cp\\u003e \\u003cem\\u003eE. histolytica\\u003c/em\\u003e cells were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 minutes at room temperature, followed by permeabilization and blocking with 0.2% saponin and 1% bovine serum albumin (BSA) in PBS for 10 minutes. Cells were incubated with primary antibodies, including anti-HA (1:200 dilution, clone 11MO; Covance, Princeton, NJ, USA.), anti-CS1 (1:500 dilution), or anti-Rab11b (1:500 dilution), for 1 hour at 4°C. After PBS washes, samples were stained with Alexa Fluor™ 488 Goat anti-Mouse IgG (H + L) and Alexa Fluor™ 594 Goat anti-Rabbit IgG (H + L) (Thermo Fisher Scientific, Waltham, MA, USA) at 1:1000 dilution, along with Hoechst 33342 (1:8000 dilution; Thermo Fisher Scientific) for 1 hour at room temperature in the dark.\\u003c/p\\u003e\\u003cp\\u003eCaco-2 monolayers, cultured for 21 days on Millicell EZ slides (Merck Millipore, Burlington, MA, USA), were co-cultured with \\u003cem\\u003eE. histolytica\\u003c/em\\u003e HA-KERP2 or GFP-HA-KERP2-expressing strains for 2 hours. Following interaction, cells were fixed with 4% PFA in PBS for 20 minutes, then permeabilized and blocked with 0.2% saponin, 1% BSA, and 50 mM glycine in PBS for 2 hours. Samples were incubated overnight at 4°C with anti-HA (1:200 dilution, clone 11MO; Covance). After washing, cells were stained with Alexa Fluor™ 488 Goat anti-Mouse IgG (H + L) (Thermo Fisher Scientific) at 1:1000 dilution, with or without Alexa Fluor™ 594 Phalloidin (1:400 dilution; Thermo Fisher Scientific), and Hoechst 33342 (1:8000 dilution; Thermo Fisher Scientific) for 1 hour at room temperature in the dark.\\u003c/p\\u003e\\u003cp\\u003e \\u003cem\\u003eE. histolytica\\u003c/em\\u003e GFP-HA-KERP2 strains were pre-stained with CellTracker Deep Red (Thermo Fisher Scientific) in Opti-MEM (Thermo Fisher Scientific) for 1 hour. Parasites were seeded onto the 3D-crypt enterocyte model and allowed to interact for 2 hours. Immunofluorescence staining followed the same protocol as for Caco-2 cells.\\u003c/p\\u003e\\u003cp\\u003eFor Caco-2 morphology observation after treatment with \\u003cem\\u003eE. histolytica\\u003c/em\\u003e transformants, the same IFA process was used. Primary antibody anti-E-cadherin (1:1000 dilution, clone 3195; Cell Signaling Technology, Danvers, MA, USA) and secondary antibody Alexa Fluor™ 488 Goat anti-Rabbit IgG (H + L) (Thermo Fisher Scientific) were used together with Alexa Fluor™ 594 Phalloidin (1:400 dilution; Thermo Fisher Scientific) and Hoechst 33342 (1:8000 dilution; Thermo Fisher Scientific).\\u003c/p\\u003e\\u003cp\\u003eImaging was performed using an FV3000 confocal microscope (EVIDENT) for \\u003cem\\u003eE. histolytica\\u003c/em\\u003e and Caco-2 cells, and a Dragonfly High-Speed Confocal Microscope System (Oxford Instruments, Abingdon, Oxfordshire, UK) for 3D-crypt enterocytes. Image processing and line intensity profile analyses were conducted using Imaris 10.2 and Fiji software (Schindelin et al., \\u003cspan citationid=\\\"CR86\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e).\\u003c/p\\u003e\\u003ch2\\u003eLive Imaging\\u003c/h2\\u003e\\u003cp\\u003eLive-cell imaging was conducted to observe the localization and dynamics of GFP-HA-tagged KERP2 constructs in real time. For imaging, cells were pre-stained with Hoechst 33342 (Thermo Fisher Scientific) in Opti-MEM (Thermo Fisher Scientific) for 40 minutes and maintained in complete BI-S33 medium at 35.5°C. Live cells were plated on glass-bottom dishes and allowed to settle for 30 minutes before imaging. Imaging was performed using an LSM 780 confocal microscope (Carl Zeiss Microscopy, White Plains, NY, USA) equipped with a temperature-controlled chamber set to 35.5°C and a 63× oil-immersion objective. Time-lapse images were captured continuously for up to 5 minutes.\\u003c/p\\u003e\\u003cp\\u003eFor KERP2 transfer studies, 21-day cultured Caco-2 monolayers established on glass-bottom dishes were pre-stained with Hoechst 33342 (Thermo Fisher Scientific) in EMEM (ATCC) for 40 minutes. GFP-HA-KERP2-expressing \\u003cem\\u003eE. histolytica\\u003c/em\\u003e was added to the Caco-2 monolayers and incubated for 30 minutes. Live imaging was performed as described above, with time-lapse images captured continuously for up to 30 minutes. Z-stack scanning was performed following time-lapse imaging.\\u003c/p\\u003e\\u003cp\\u003eImage acquisition and analysis were conducted using Imaris 10.2 (Oxford Instruments) and Fiji software.\\u003c/p\\u003e\\u003ch2\\u003eImmuno-electron Microscopy\\u003c/h2\\u003e\\u003cp\\u003eTrophozoites overexpressing HA-KERP2 and HA-mock were cultured in BIS medium and incubated with gold disks at 35.5°C overnight to facilitate attachment. The disks carrying attached amoebae were rapidly frozen in liquid propane at − 175°C. Freeze substitution was carried out in 2% tannic acid in ethanol with 2% distilled water at − 80°C for 48 hours. The samples were then gradually warmed to − 20°C for 4 hours and subsequently to 4°C for 1 hour. Dehydration was performed with three changes of anhydrous ethanol at 4°C for 30 minutes each. Dehydrated samples were infiltrated with a 50:50 mixture of ethanol and resin (LR White; London Resin Co. Ltd., Berkshire, UK) at 4°C for 30 minutes, followed by three changes of 100% LR White resin at 4°C for 30 minutes each. Samples were transferred to fresh 100% resin and polymerized at 50°C overnight. Polymerized samples were sectioned into ultra-thin slices (70 nm) using a diamond knife on an ultramicrotome (Ultracut UCT; Leica Microsystems, Vienna, Austria). Sections were mounted on nickel grids and incubated overnight at 4°C with a primary antibody (anti-HA) diluted in 1% BSA/PBS. Grids were washed three times with 1% BSA/PBS for 1 minute each, then incubated for 2 hours at room temperature with a secondary antibody conjugated to 15 nm gold particles (goat anti-mouse IgG pAb). After secondary antibody incubation, grids were washed with PBS and fixed with 2% glutaraldehyde in 0.1 M phosphate buffer. Finally, grids were dried and stained with 2% uranyl acetate for 10 minutes, followed by lead stain solution (Sigma-Aldrich Co., Tokyo, Japan) at room temperature for 3 minutes. Samples were observed using a transmission electron microscope (JEM-1400Plus; JEOL Ltd., Tokyo, Japan) operating at an acceleration voltage of 100 kV. Digital images (3296 × 2472 pixels) were captured using a CCD camera (EM-14830RUBY2; JEOL Ltd., Tokyo, Japan).\\u003c/p\\u003e\\u003ch2\\u003eqRT-PCR and RNA-seq\\u003c/h2\\u003e\\u003cp\\u003eTotal RNA was extracted from KERP2gs or psAP-mock trophozoites, and complementary DNA (cDNA) was synthesized as described in previous methods. The efficiency of KERP2 silencing in trophozoites was validated using quantitative real-time PCR (qRT-PCR). RNA polymerase II (EHI_056690) was used as the internal control. Primers were designed to amplify a 493 bp segment of KERP2 and a 204 bp segment of RNA polymerase II. Twenty-fold diluted cDNA from each strain was used as a template in reactions performed with the Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), following the manufacturer’s protocol. qRT-PCR was conducted on a StepOne Plus Real-Time PCR System (Applied Biosystems), and data analysis was performed using the DataAssist software (Thermo Fisher Scientific). RNA-seq was performed an Illumina NovaSeq 6000 platform (Fujifilm, Japan) similarly as described below.\\u003c/p\\u003e\\u003cp\\u003eFor co-culture experiments, total RNA was extracted from Caco-2 cells interacting with HA-KERP2-expressing \\u003cem\\u003eE. histolytica\\u003c/em\\u003e G3 strain, psAP-KERP2gs trophozoites, or G3 control trophozoites after 4 hours of co-culture in complete EMEM medium. RNA integrity was assessed using an Agilent 2100 Bioanalyzer, and sequencing libraries were prepared using a strand-specific RNA-seq library preparation kit. Libraries were sequenced on an Illumina NovaSeq X Plus (Novogene, China), generating paired end reads. Sequence reads were quality-checked using FastQC (v0.11.8) and trimmed using Trimmomatic (v0.39) (Bolger et al., \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). Trimmed reads were aligned to the \\u003cem\\u003eE. histolytica\\u003c/em\\u003e reference genome assembly (AmoebaDB-68) or the human reference genome (GRCh38.p14) using STAR (v2.7.10a) (Dobin et al., \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e) with default parameters and gene annotation provided in the corresponding GTF file. Mapped reads were indexed and sorted using Samtools (v1.15.1) (Danecek et al., \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Read count quantification at the gene level was performed using FeatureCounts (v2.0.1) (Liao et al., \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e) with strand-specific parameters. Differential gene expression analysis was conducted using the DESeq2 R package (v1.36.0) (Love et al., \\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e), with three biological replicates per condition. Gene expression normalization was performed using DESeq2’s median-of-ratios method. Log2 fold changes and adjusted P-values were computed using the Benjamini-Hochberg method to control the false discovery rate (FDR). Genes with an adjusted P-value (padj) ≤ 0.05 and an absolute log2 fold change ≥ 1 were classified as differentially expressed. Principal component analysis (PCA) and hierarchical clustering were performed to assess sample variance. Volcano plots and heatmaps were generated using ggplot2 (v3.5.1) (Wickham and SpringerLink (Online service), 2016), ggrepel (v0.9.5) (Slowikowski, \\u003cspan citationid=\\\"CR93\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e), and pheatmap (v1.0.12) (Kolde, \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e) to visualize differentially expressed genes across conditions. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted using clusterProfiler (v4.2.2) (Stirling et al., \\u003cspan citationid=\\\"CR97\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e) or ShinyGO (v0.82) (Ge et al., \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e) to identify functional categories associated with differentially expressed genes.\\u003c/p\\u003e\\u003ch2\\u003eGrowth Kinetics\\u003c/h2\\u003e\\u003cp\\u003eApproximately 3 × 10⁴ exponentially growing trophozoites of the \\u003cem\\u003eE. histolytica\\u003c/em\\u003e G3 strain, transformed with either psAP-KERP2gs or psAP-mock (control), were inoculated into 6 mL of fresh BI-S-33 medium supplemented with 10 µg/mL G418. The cultures were incubated under standard conditions, and trophozoites were counted every 24 hours using a hemocytometer to assess growth over time.\\u003c/p\\u003e\\u003ch2\\u003eCysteine Protease (CP) Activity Assay\\u003c/h2\\u003e\\u003cp\\u003eApproximately 1.2 × 10⁶ \\u003cem\\u003eE. histolytica\\u003c/em\\u003e transformants were cultured in 500 µL of Transfection Medium (Opti-MEM supplemented with 5 mg/mL L-cysteine and 1 mg/mL ascorbic acid) at 35.5°C for 1 hour. After incubation, cells were aliquoted into 50 µL samples and centrifuged at 3000 × g for 3 minutes. The supernatant was subsequently centrifuged at 13,000 × g for 5 minutes to collect the released form of cysteine protease (CP) activity. The cell pellet was lysed in 100 µL of PBS using three freeze-thaw cycles, and cellular debris was removed by centrifugation at 15,000 × g for 5 minutes. The resulting supernatant from this step was collected as the intracellular form of CP activity. Both released and intracellular samples were pre-incubated in assay buffer (0.1 M KH₂PO₄, pH 6.1, 1 mM EDTA, 2 mM DTT) at room temperature for 15 minutes to activate pro-forms of CP. Following activation, 75 µL of the enzyme mixture was combined with Benzyloxycarbonyl-L-arginyl-L-arginine 4-methylcoumaryl-7-amide (#3123-v, Peptide Institute, Osaka, Japan) to achieve a final substrate concentration of 10 mM. Fluorescence emission at 460 nm (excitation at 355 nm) was measured for 1 hour using a SpectraMax Paradigm multimode microplate reader (Molecular Devices, San Jose, CA, USA). 7-amino-4-methylcoumarin (#3099-v, Peptide Institute) was used as a standard to quantify specific CP activities. Results were expressed as fluorescence intensity corresponding to the production of 4-methylcoumaryl-7-amide per milligram of lysate protein. Statistical significance was evaluated using two-way ANOVA.\\u003c/p\\u003e\\u003ch2\\u003eNuclear Fractionation and Co-immunoprecipitation\\u003c/h2\\u003e\\u003cp\\u003eNuclear protein fractions were prepared from approximately 2 × 10⁷ cells of \\u003cem\\u003eE. histolytica\\u003c/em\\u003e HA-KERP2, HA-KERP2\\u003csup\\u003e∆185–239\\u003c/sup\\u003e, and HA-mock strains. Prior to nuclear fractionation, cells were cross-linked with 0.8 mg/mL Pierce dithiobis (succinimidyl propionate) (DSP) (Thermo Fisher Scientific) in PBS to stabilize protein-protein interactions. Nuclear protein extraction was conducted using the Nuclear Complex Co-IP Kit (#54001, Active Motif, Carlsbad, CA, USA) according to the manufacturer’s protocol. Extracted nuclear fractions were treated with the enzymatic cocktail reagent included in the kit, followed by nucleic acid digestion to degrade DNA and RNA, ensuring a focus on protein-protein interactions. The nuclear extracts were diluted in the IP low buffer provided in the kit, supplemented with protease and phosphatase inhibitors. Cytosolic fractions were also prepared for comparative analysis. Diluted nuclear and cytosolic fractions were incubated separately with anti-HA antibody (clone 11MO, Covance) overnight at 4°C. Antibody-bound complexes were captured by incubating the samples with 45 µL of pre-washed Dynabeads™ Protein A (Thermo Fisher Scientific) for 1 hour at 4°C. To minimize non-specific interactions, beads were washed three times with 500 µL of IP low buffer containing BSA, followed by three additional washes with 500 µL of IP low buffer without BSA. Bound proteins were eluted by incubating the beads with 0.3 mg/mL HA peptide (#I2149, Sigma-Aldrich) overnight at 4°C. Eluted samples were collected for downstream phosphorylation validation, western blot analysis, or mass spectrometry-based identification.\\u003c/p\\u003e\\u003ch2\\u003eExtracellular Vesicle Isolation\\u003c/h2\\u003e\\u003cp\\u003eApproximately 50 mL of serum-deprived culture medium from \\u003cem\\u003eE. histolytica\\u003c/em\\u003e transformants was collected and centrifuged at 1,000 × g for 10 minutes at 4°C to remove intact cells. The resulting supernatant was filtered through a 0.22 µm membrane using a 50 mL syringe to eliminate larger debris. The filtered medium was then concentrated using a 100 kDa MWCO Amicon filter (Merck Millipore) at 4,000 × g for 15 minutes at 4°C. The concentrated cell-free medium was transferred to an ultracentrifuge tube and subjected to ultracentrifugation at 100,000 × g for 75 minutes at 4°C to pellet extracellular vesicles (EVs), including exosomes. The EV pellet was washed with 1× PBS, followed by a repeat of the ultracentrifugation step under identical conditions. After discarding the supernatant, the final EV pellet was resuspended in 50 µL of 1× PBS. For EV lysis, 1% Triton X-100 was added to the resuspended pellet, and the mixture was incubated on ice for 30 minutes.\\u003c/p\\u003e\\u003ch2\\u003ePreparation of Recombinant Proteins\\u003c/h2\\u003e\\u003cp\\u003ePlasmids pET-KERP2, pET151-GFP-KERP2, and pET151-GFP, designed to express recombinant His-GFP-KERP2 and His-GFP proteins, respectively, were introduced into \\u003cem\\u003eE. coli\\u003c/em\\u003e BL21 (DE3) cells by heat shock at 42°C for 1 minute. Transformed \\u003cem\\u003eE. coli\\u003c/em\\u003e cells were initially grown in 100 mL of Luria Bertani (LB) medium supplemented with 100 µg/mL carbenicillin at 37°C. The overnight culture was then used to inoculate 500 mL of fresh LB medium, and the cells were grown at 37°C with shaking at 200 rpm. When the optical density at 600 nm (A600) reached 0.4, the cultures were chilled on ice, and 2% ethanol and 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) were added to induce protein expression. Cultivation was continued for an additional 16 hours at 18°C. Induced \\u003cem\\u003eE. coli\\u003c/em\\u003e cells were harvested by centrifugation at 8,000 × g for 10 minutes at 4°C. The cell pellets were washed with Tris-saline buffer (10 mM Tris-HCl, pH 7.6, and 150 mM NaCl) and resuspended in 40 mL of lysis buffer containing 50 mM Tris-HCl (pH 7.6), 250 mM NaCl, 10% (w/v) sucrose, 100 µg/mL lysozyme, 0.5 µg/mL E64, 1× cOmplete Mini Protease Inhibitor Cocktail (Roche), and 1 mM phenylmethylsulfonyl fluoride (PMSF). The suspension was incubated at room temperature for 20 minutes and lysed using a French press. Triton X-100 was added to a final concentration of 0.1% (v/v), and the lysate was centrifuged at 25,000 × g for 30 minutes at 4°C to remove cell debris. The clarified supernatant was incubated with 1 mL of 50% Ni²⁺-NTA His-Bind resin slurry at 4°C for 1 hour with gentle rotation. The resin was transferred to a column and washed three times with wash buffer containing 50 mM Tris-HCl (pH 7.6) and 2 M KCl. Bound proteins were eluted using elution buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 10% (v/v) glycerol, and 10–750 mM imidazole. The integrity and purity of the recombinant proteins were confirmed by 12% SDS-PAGE, followed by Coomassie Brilliant Blue staining or western blot analysis.\\u003c/p\\u003e\\u003ch2\\u003eProtein–lipid overlay assays\\u003c/h2\\u003e\\u003cp\\u003eThe soluble fraction of recombinant His-KERP2 or His-GFP was obtained as described above and used to probe a P-6001 phospholipid membrane strip (Echelon Biosciences, Salt Lake City, UT, USA). The membrane was blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 2 hours at room temperature (RT) before incubation with 1 µg/mL of recombinant protein in PBS containing 3% BSA for 16 hours at 4°C. Following incubation, the membrane was washed three times with PBS containing 0.1% Tween-20 (PBS-T) and then incubated with an anti-His primary antibody (His-Tag (27E8) Mouse mAb #2366, Cell Signaling Technology, 1:1000) in PBS with 3% BSA for 2 hours at RT. After additional washes, the membrane was incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (Cat# 32430, Invitrogen, Thermo Fisher Scientific, 1:5000) in PBS with 3% BSA for 1 hour at RT. The blot was washed, developed using a chemiluminescent HRP substrate (Merck Millipore), and visualized using a ChemiDoc™ Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).\\u003c/p\\u003e\\u003ch2\\u003eSample Preparation for KERP2 Translocation in Caco-2 Cells\\u003c/h2\\u003e\\u003cp\\u003eConfluent Caco-2 cells cultured in T75 flasks (~ 5 × 10⁶ cells per flask) were co-cultured with \\u003cem\\u003eE. histolytica\\u003c/em\\u003e HA-KERP2 strains at a 5:1 ratio (Caco-2: \\u003cem\\u003eE. histolytica\\u003c/em\\u003e) for 1, 2, and 3 hours in complete EMEM (ATCC). For control experiments, \\u003cem\\u003eE. histolytica\\u003c/em\\u003e HA-mock strains were co-cultured with Caco-2 cells for 1 hour. An additional experimental group involved pretreatment of \\u003cem\\u003eE. histolytica\\u003c/em\\u003e HA-KERP2 strains with 2% galactose in complete Diamond’s BI-S-33 medium for 30 minutes prior to co-culture. During the interaction for this group, 2% galactose was also added to the complete EMEM for a 1-hour incubation. After co-culture, the mixtures were washed three times with pre-cooled 2% galactose in PBS (Thermo Fisher Scientific) to remove \\u003cem\\u003eE. histolytica\\u003c/em\\u003e cells. The remaining Caco-2 cells were collected, while \\u003cem\\u003eE. histolytica\\u003c/em\\u003e cells were processed separately. Both cell types were lysed using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific), supplemented with cOmplete™ Protease Inhibitor Cocktail (Roche). The lysates were subjected to western blotting for the detection of KERP2 translocation. Anti-HA antibody (ab9110, Abcam, Cambridge, UK) was used to enhance detection sensitivity during western blot analysis.\\u003c/p\\u003e\\u003ch2\\u003eCaco2 Co-immunoprecipitation\\u003c/h2\\u003e\\u003cp\\u003eConfluent Caco-2 cells grown in T75 flasks (~ 5 × 10⁶ cells per flask) were used to interact with \\u003cem\\u003eE. histolytica\\u003c/em\\u003e HA-KERP2 or HA-mock strains at a 5:1 ratio (Caco-2: \\u003cem\\u003eE. histolytica\\u003c/em\\u003e) for 1 hour. Following interaction, six flasks of co-cultures were washed three times with pre-cooled 2% galactose in PBS to remove \\u003cem\\u003eE. histolytica\\u003c/em\\u003e cells. The remaining Caco-2 cells were harvested using a cell scraper. The collected Caco-2 cells were cross-linked with 0.8 mg/mL Pierce DSP (ThermoFisher Scientific) in PBS for stabilization of protein-protein interactions. After cross-linking, cells were lysed using M-PER Mammalian Protein Extraction Reagent (ThermoFisher Scientific) supplemented with a cOmplete protease inhibitor cocktail (Roche). Lysates were incubated with anti-HA antibody (clone 11MO; Covance) overnight at 4°C. The antibody-bound complexes were captured by incubation with 40 µL of pre-washed Dynabeads Protein A (Invitrogen, ThermoFisher) for 1 hour at 4°C. Beads were washed three times with Washing Buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, 0.1% BSA) and three additional times with Washing Buffer lacking BSA to minimize non-specific interactions. Bound proteins were eluted by incubating the beads with 0.3 mg/mL HA peptide (Sigma-Aldrich) overnight at 4°C. Eluted samples were collected and prepared for downstream analyses.\\u003c/p\\u003e\\u003ch2\\u003eFlamingo Gel Staining\\u003c/h2\\u003e\\u003cp\\u003eCo-IP samples were separated by SDS-PAGE as described in the relevant methods sections. Following electrophoresis, gels were subjected to Flamingo fluorescent gel staining (Bio-Rad Laboratories) to visualize protein elution in nuclear co-IP and Caco-2 co-IP pull-down samples. The staining was performed according to the manufacturer’s protocol. Stained gels were imaged using a ChemiDoc imaging system (Bio-Rad Laboratories) to detect protein bands.\\u003c/p\\u003e\\u003ch3\\u003eWestern Blotting\\u003c/h3\\u003e\\u003cp\\u003eProtein samples were separated by SDS-PAGE or Phos-tag™ SDS-PAGE (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and transferred onto PVDF membranes (Merck Millipore) using a semi-dry transfer system. Membranes were blocked with 5% non-fat milk in TBST (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) for 30 minutes at room temperature. Blocked membranes were incubated overnight at 4°C with primary antibodies, including anti-HA (1:1000, clone 11MO, BioLegend, San Diego, CA, USA), anti-CS1 (1:1000, in-house source), anti-GAPDH (1:5000, AM4300, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), anti-Histone H3 (1:1000, ab1791, Abcam), or anti-His (1:1000, #2366, Cell Signaling Technology, Danvers, MA, USA). After primary antibody incubation, membranes were washed three times with TBST and then incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Secondary antibodies used included Donkey anti-Rabbit IgG (H + L) Cross-Adsorbed, HRP (31458, Invitrogen, Thermo Fisher Scientific) and Goat anti-Mouse IgG (H + L), HRP (32430, Invitrogen, Thermo Fisher Scientific). Following secondary antibody incubation, membranes were washed three more times with TBST. Signals were detected using a chemiluminescence HRP substrate (Merck Millipore) and visualized using a ChemiDoc™ Imaging System (Bio-Rad Laboratories).\\u003c/p\\u003e\\u003ch2\\u003eMass Spectrometry\\u003c/h2\\u003e\\u003cp\\u003eProtein extracts were buffer-exchanged using SP3 paramagnetic beads (Cytiva, Marlborough, MA, USA). Samples were rehydrated in 100 µL of 10 mM triethylammonium bicarbonate (TEAB) containing 1% SDS. Disulfide bonds were reduced with 10 µL of 50 mM dithiothreitol (DTT) at 60°C for 1 hour. After cooling to room temperature (RT), the pH was adjusted to approximately 7.5, and alkylation was performed by adding 10 µL of 100 mM iodoacetamide (Sigma-Aldrich) and incubating in the dark at RT for 15 minutes. SP3 beads (100 µg; 2 µL of 50 µg/µL) were added to the samples, followed by the addition of 120 µL of 100% ethanol. Samples were incubated at RT with shaking for 5 minutes to bind proteins to the beads. Beads were then washed three times with 180 µL of 80% ethanol. On-bead protein digestion was performed overnight at 37°C using trypsin (Pierce Trypsin, Thermo Fisher Scientific, Waltham, MA, USA; 1 µg enzyme per sample). After digestion, the supernatant was removed, dried, and rehydrated in 2% acetonitrile/0.1% formic acid for mass spectrometry analysis. Peptide analysis was performed by reverse-phase chromatography-tandem mass spectrometry (LC-MS/MS) using an EasyLC 1100 UPLC system interfaced with an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a 75 µm × 15 cm column (PicoFrit Self-pack emitter, New Objective, Woburn, MA, USA) packed in-house with ReproSIL-Pur-120-C18-AQ resin (3 µm, 120 Å bulk phase, Dr. Maisch GmbH, Ammerbuch, Germany). Separation was achieved using a 0–100% acetonitrile gradient in 0.1% formic acid over 90 minutes at a flow rate of 300 nL/minute. Survey scans of precursor ions were acquired over a range of 350–1800 m/z with a resolution of 120,000 at 200 m/z, an automatic gain control (AGC) target of 4 × 10⁵, and an RF lens setting of 45%. Internal mass calibration was used for accuracy. Precursor ions were individually isolated within a 1.5 m/z window using data-dependent acquisition with a 15-second dynamic exclusion. Fragmentation was performed using higher-energy collision dissociation (HCD) at a collision energy of 30. Fragment ions were analyzed at a resolution of 30,000 with an AGC target of 1 × 10⁵. The raw data files were analyzed using Mascot software (Matrix Science, London, UK; version 2.8.2) with the UniProt \\u003cem\\u003eE. histolytica\\u003c/em\\u003e database (taxon ID: 294381; 20,546 entries). Searches assumed trypsin as the digestion enzyme, with a fragment ion mass tolerance of 10.0 ppm and a parent ion mass tolerance of 5.0 ppm. Carbamidomethylation of cysteine was specified as a fixed modification, while deamidation of asparagine and glutamine, oxidation of methionine, phosphorylation of serine, threonine, and lysine, and formylation of lysine and the protein N-terminus were defined as variable modifications. Peptide identifications were validated and processed using Scaffold software (Proteome Software Inc., Portland, OR, USA). PeptideProphet was used for peptide validation with a 1% false discovery rate (FDR), while ProteinProphet was used for protein inference with a confidence threshold of 95%.\\u003c/p\\u003e\\u003ch2\\u003eEdU Incorporation Assay\\u003c/h2\\u003e\\u003cp\\u003eFour-day-cultured Caco-2 cells were co-cultured with 1 × 10⁴ \\u003cem\\u003eE. histolytica\\u003c/em\\u003e transformants at a ratio of approximately 10:1 (Caco-2: \\u003cem\\u003eE. histolytica\\u003c/em\\u003e) for 2 hours. After interaction, the co-cultures were washed three times with pre-cooled 2% galactose in PBS to remove \\u003cem\\u003eE. histolytica\\u003c/em\\u003e transformants. To assess cell proliferation, 15 µM EdU was incorporated into the Caco-2 cells for 6 hours. Staining was performed using the Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 488 dye (Thermo Fisher Scientific), according to the manufacturer’s instructions.\\u003c/p\\u003e\\u003cp\\u003eEdU staining was analyzed using CellProfiler 4.2.7 (Stirling et al., \\u003cspan citationid=\\\"CR97\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e) to quantify cell proliferation in Caco-2 cells cocultured with \\u003cem\\u003eE. histolytica\\u003c/em\\u003e transformants. Images of EdU-labeled nuclei (Alexa Fluor™ 488) and Hoechst 33342-stained nuclei were processed to identify EdU-positive cells. Hoechst-stained nuclei were segmented as primary objects based on intensity thresholds, with object size constrained to 30–100 pixels to exclude artifacts. EdU-positive regions were identified as secondary objects using propagation-based segmentation linked to the primary nuclei. Illumination correction was applied to normalize intensity across EdU-stained images. Morphological measurements were extracted, including the proportion of EdU-positive cells relative to the total nuclei. Data from five images per condition were exported and statistically analyzed. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed using GraphPad Prism (version 10.2.0 MacOS, GraphPad Software, Boston, Massachusetts USA).\\u003c/p\\u003e\\u003ch2\\u003eMorphological Analysis\\u003c/h2\\u003e\\u003cp\\u003eCaco-2 cells cocultured with \\u003cem\\u003eE. histolytica\\u003c/em\\u003e transformants were imaged as described in the IFA method above. Morphological analysis was conducted using CellProfiler 4.2.7. For each treatment, 5 images of Hoechst 33342-stained nuclei, E-cadherin-stained cell boundaries, and Phalloidin-stained F-actin were analyzed to quantify changes in cellular morphology. Nuclei were identified as primary objects using intensity-based segmentation of DNA-stained images, with a size range of 30–100 pixels. Cell boundaries were segmented as secondary objects using propagation-based methods, expanding from the nuclei and leveraging both E-cadherin and Phalloidin images for boundary definition. Illumination correction was applied to E-cadherin and Phalloidin images to normalize intensity variations. Data were exported as .csv files for statistical analysis, with parameters for segmentation and measurement optimized to ensure accurate object identification and quantification. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed using GraphPad Prism (version 10.2.0 MacOS, GraphPad Software).\\u003c/p\\u003e\\u003cp\\u003eMorphological measurements were calculated, including:\\u003c/p\\u003e\\u003cp\\u003eForm Factor, defined as \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\text{Form:Factor}=\\\\frac{4{\\\\pi:}A}{{P}^{2}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e, where A is the area and P is the perimeter of the cell, used to assess circularity.\\u003c/p\\u003e\\u003cp\\u003eAspect Ratio, defined as \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\text{Aspect:Ratio}=\\\\frac{\\\\text{Major:Axis}}{\\\\text{Minor:Axis}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e, representing the elongation of cells.\\u003c/p\\u003e\\u003ch2\\u003eTransepithelial Electrical Resistance (TEER) Measurement\\u003c/h2\\u003e\\u003cp\\u003eTEER was measured to assess epithelial barrier integrity using the EVOM™ Manual for TEER Measurement system (World Precision Instruments, Sarasota, FL, USA). Caco-2 cells were cultured on 12 mm Transwell inserts with a 0.4 µm pore size (3460, Corning Inc., Corning, NY, USA) as described in the cell culture method. Cells were maintained for 21 days until the TEER value stabilized at approximately 1300 Ω, indicating the formation of a confluent monolayer. Caco-2 cells were interacted with \\u003cem\\u003eE. histolytica\\u003c/em\\u003e transformants at a ratio of 10:1 (Caco-2: \\u003cem\\u003eE. histolytica\\u003c/em\\u003e) or different concentration of recombinant proteins. TEER measurements were recorded before the interaction and at 1-hour intervals for up to 4 hours. Data at each time point were normalized to the baseline TEER value measured before interaction (time 0, set as 100%). Statistical significance at each time point was determined using a two-way ANOVA follwed by Dunnett’s multiple comparisons test on GraphPad Prism (version 10.2.0 MacOS, GraphPad Software).\\u003c/p\\u003e\\u003ch3\\u003eWound Healing Assay\\u003c/h3\\u003e\\u003cp\\u003eWound healing assay was conducted using Culture-Insert 2 Well in µ-Dish 35 mm (81176, ibidi GmbH, Gräfelfing, Germany) to create a Caco-2 monolayer with a defined gap. Caco-2 cells were cultured for 4 days to establish a confluent monolayer. \\u003cem\\u003eE. histolytica\\u003c/em\\u003e transformants (2 × 10³) were seeded on either side of the insert and allowed to interact with the Caco-2 monolayer for 2 hours. Following interaction, \\u003cem\\u003eE. histolytica\\u003c/em\\u003e was removed by washing with pre-cooled 2% galactose in PBS. To minimize the effects of cell proliferation, the Caco-2 monolayer was cultured in EMEM with 2% FBS after washing. Images of the gap area were captured at 1-hour intervals for up to 24 hours using an optical microscope, with 2–3 positions imaged per treatment group. Gap areas (width pixels) at each time point were measured using the Manual Wound Healing Size Tool plugin in Fiji software (Suarez-Arnedo et al., \\u003cspan citationid=\\\"CR98\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). Recovery rates were determined by linear regression analysis of gap closure over time. Statistical significance of differences in recovery rates (slopes) between treatments was assessed using simple linear regression in GraphPad Prism (version 10.2.0 MacOS, GraphPad Software).\\u003c/p\\u003e\\u003ch3\\u003eFlow Cytometry\\u003c/h3\\u003e\\u003cp\\u003eFlow cytometry was performed to analyze Caco-2 cells treated with recombinant His-GFP-KERP2 or His-GFP proteins. Caco-2 cells were cultured for 21 days as described above and treated with 3 µM of His-GFP-KERP2 or His-GFP under various conditions. After treatment, cells were washed three times with 1× PBS and incubated with 0.5% trypsin for 5 minutes to remove excess recombinant protein and isolate the Caco-2 cells. The trypsinized cells were directly loaded into a BD Accuri™ C6 Plus flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) at a low flow rate. Data acquisition was performed using the instrument’s standard settings, and subsequent analysis and plotting were carried out using FlowJo software (BD Biosciences) and GraphPad Prism.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eStatistical information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eqRT-PCR:\\u0026nbsp;Statistical differences were determined by multiple unpaired t-tests.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eGrowth kinetics: Statistical differences at individual time points were assessed using two-way ANOVA (n=3).\\u003c/p\\u003e\\n\\u003cp\\u003eFlow cytometry: Data acquisition was performed using a BD Accuri\\u0026trade; C6 Plus flow cytometer (BD Biosciences), and subsequent analysis of median fluorescence intensity and plotting were carried out using FlowJo software (BD Biosciences). Statistical differences of MFI at individual time points were assessed using two-way ANOVA (n=3).\\u003c/p\\u003e\\n\\u003cp\\u003eCysteine protease (CP) activity assay: Statistical differences were evaluated using one-way ANOVA (n=8).\\u003c/p\\u003e\\n\\u003cp\\u003eEdU incorporation assay: Statistical significance for quantifying EdU-positive cells was determined using one-way ANOVA followed by Dunnett\\u0026rsquo;s multiple comparisons test.\\u003c/p\\u003e\\n\\u003cp\\u003eMorphological analysis: Form factor and aspect ratio were computed, and statistical significance was determined using one-way ANOVA followed by Dunnett\\u0026rsquo;s multiple comparisons test.\\u003c/p\\u003e\\n\\u003cp\\u003eWound healing assay: Recovery rates were determined by linear regression analysis of gap closure over time, and statistical significance of differences in recovery rates (slopes) between treatments was assessed using simple linear regression.\\u003c/p\\u003e\\n\\u003cp\\u003eTransepithelial Electrical Resistance (TEER) Measurement: Measurements were recorded at various time points, and statistical significance was determined using two-way ANOVA followed by Dunnett\\u0026rsquo;s multiple comparisons test.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eRNA-seq data have been deposited at GEO: GSE290785 and GSE290901. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported partly by Grants-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS) (JP21H02723 to T.N.), Fostering Joint International Research (B) from JSPS (JP21KK135 to T.N.), and Grant for Science and Technology Research Partnership for Sustainable Development (SATREPS) from Japan Agency for Medical Research and Development (AMED) and Japan International Cooperation Agency (JICA) (JP24jm0110022) to T.N., Grant for research on emerging and re-emerging infectious diseases from AMED (JP24fk0108680 to T.N.), and support from the University of Tokyo Pandemic Preparedness, Infection, and Advanced Research Center (UTOPIA) and AMED (JP243fa627001) to T.N.. This work was also partly supported by JSPS Grants-in-Aid for Scientific Research (23K06514 to H.J.S.), JSPS Bilateral Joint Research Grant (JPJSBP120223203 to H.J.S.)., and JST SPRING (Grant Number JPMJSP2108 to R.P.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank to Dr. Cecilia Villegas Novoa, Dr. Yuli Wang, and Prof. Nancy Allbritton from the Department of Bioengineering, University of Washington and Dr. Soichiro Ishihara and Dr. Yuzo Nagai from the Department of Surgery, University of Tokyo Hospital for their help in constructing 3D-crypt model. We thank to Dr. ISHINO Tomoko and Dr. Sinzawa Naoki from the Department of Parasitology and Tropical Medicine, Science Tokyo for their help in optimizing ChIP protocol.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eR.P., H.S. and T.N. contributed to conceptualization; R.P., H.S. contributed to methodology; R.P. contributed to investigation and experiments; R.P., H.S. and T.N. contributed to manuscript writing.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNo declaration of interests.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMaterials \\u0026amp; correspondence\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTomoyoshi Nozaki (nozaki@m.u-tokyo.ac.jp) is addressed for correspondence and material requests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAnsart S, Perez L, Vergely O, Danis M, Bricaire F, Caumes E (2005) Illnesses in travelers returning from the tropics: a prospective study of 622 patients. 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In StatPearls\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-portfolio\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Nature Portfolio\",\"twitterHandle\":\"\",\"acdcEnabled\":false,\"dfaEnabled\":false,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Entamoeba histolytica, KERP2, virulence factor, host-pathogen interaction, cytoskeletal remodeling, cysteine proteases, epithelial barrier integrity, amoebiasis\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6191032/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6191032/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e \\u003cem\\u003eEntamoeba histolytica\\u003c/em\\u003e, the protozoan parasite responsible for amoebiasis, deploys a complex array of virulence factors to establish infection and evade host defenses. Here, we identify KERP2 as a dual-function effector that regulates both parasite homeostasis and host cell remodeling. Bioinformatic analyses, cellular localization assays, and functional studies show that KERP2 localizes to the parasite nucleus, associates with chromatin, and modulates transcription, particularly regulating cysteine protease expression and sulfur metabolism. Concurrently, KERP2 is translocated into host epithelial cells, where it manipulates the G1/S transition, interacts with cytoskeletal regulators, and promotes actin remodeling, ultimately compromising epithelial barrier function. Our results elucidate how \\u003cem\\u003eE. histolytica\\u003c/em\\u003e harnesses KERP2 to coordinate intracellular processes in the parasite while orchestrating pathogenic alterations in host cells. These insights shed light on a broader mechanism by which extracellular pathogens deploy multifunctional effectors to optimize virulence and adapt to diverse host environments, providing a valuable framework for studies on pathogen-host interactions beyond amoebiasis.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\",\"manuscriptTitle\":\"Dual Role of Entamoeba histolytica KERP2 in Regulating Gene Expression and Modulating Host Cell Function for Intestinal Colonization\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-04-28 06:02:06\",\"doi\":\"10.21203/rs.3.rs-6191032/v1\",\"editorialEvents\":[],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-communications\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"NCOMMS\",\"sideBox\":\"Learn more about [Nature Communications](http://www.nature.com/ncomms/)\",\"snPcode\":\"\",\"submissionUrl\":\"https://mts-ncomms.nature.com/\",\"title\":\"Nature Communications\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Nature Communications\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"9d72c204-4b77-411e-a77f-08cc470fd1e9\",\"owner\":[],\"postedDate\":\"April 28th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[{\"id\":46516103,\"name\":\"Biological sciences/Microbiology/Pathogens\"},{\"id\":46516104,\"name\":\"Biological sciences/Microbiology/Parasitology\"}],\"tags\":[],\"updatedAt\":\"2026-03-03T13:10:52+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-04-28 06:02:06\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6191032\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6191032\",\"identity\":\"rs-6191032\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}