IucA gene contributes to the infectivity of hypervirulent Klebsiella pneumoniae in Caco-2 cell models

IucA gene contributes to the infectivity of hypervirulent Klebsiella pneumoniae in Caco-2 cell models | 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 Research Article IucA gene contributes to the infectivity of hypervirulent Klebsiella pneumoniae in Caco-2 cell models Tingting Zhi, Chunjie Zhu, Caiqin Zi, Jinxiu Xie, Ruijun Lao, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7689359/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hypervirulent Klebsiella pneumoniae (hvKP) causes lethal systemic infections following intestinal colonization, but the mechanisms underlying its translocation across the intestinal barrier remain unclear. In this study, we used Caco-2 cells as a model to investigate the role of the aerobactin siderophore-encoding gene iucA in the invasion process of hvKP. We observed that the wild-type (WT) hvKP strain exhibited significantly higher invasion rates than the iucA -deletion mutant ( ΔiucA ) at 2 and 4 hours post-infection (P < 0.05). Furthermore, only the WT strain penetrated dense Caco-2 monolayers, indicating that iucA is essential for traversing the intestinal mucosal barrier. Interaction transcriptomics revealed distinct host-pathogen dynamics: hvKP infection significantly activated pathways related to protein export and the bacterial secretion system; Caco-2 cells upregulated pentose/glucuronate interconversions, peroxisome activity, and TGF-β signaling but downregulated protein metabolism and autophagy; The transcriptional profile of Caco-2 monolayers was minimally altered by the presence or absence of iucA , suggesting that aerobactin’s role is primarily bacterial-centric. This study reveals the central role of iucA -mediated aerobactin synthesis in the invasion of the intestinal mucosal barrier by hvKP, providing important insights into the interaction between hvKP and intestinal epithelial cells. Hypervirulent Klebsiella pneumoniae Caco-2 infection siderophore interaction transcriptomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Hypervirulent Klebsiella pneumoniae (hvKP) is a highly pathogenic subgroup of K. pneumonia , possessing a higher virulence than classical K. pneumoniae (cKP). This strain can cause invasive infections even in healthy individuals and is associated with high mortality rates (4.5%–31%) and severe sequelae (Lan et al ., 2020). Studies indicate that hvKP frequently colonizes the human intestinal mucosa, and its translocation from the gut into the bloodstream serves as a major route for organ and systemic infections (Fung et al ., 2012; Liu & Guo, 2019; Nguyen et al ., 2024). However, the precise molecular mechanisms by which hvKP breaches the intestinal mucosal barrier remain unclear. Iron acquisition systems are critical virulence determinants in hvKP, facilitating bacterial proliferation through sequestration of host iron sources. Compared to cKP, hvKP demonstrates significantly enhanced (6- to 10-fold) production of various siderophores, including enterobactin, salmochelin, aerobactin, and yersiniabactin (Kocsis, 2023; Lee et al ., 2017). This elevated siderophore production not only improves hvKP's adaptation to host environments but also enables its survival and replication in iron-restricted niches such as blood and ascites(Spellberg et al ., 2011). Notably, aerobactin - encoded by the iucABCD gene cluster and constituting over 80% of total hvKP siderophores - emerges as a pivotal virulence factor mediating systemic infection and serves as a molecular hallmark of hvKP strains(Choby, et al ., 2020; Russo et al ., 2014). In the aerobactin biosynthesis pathway of hvKP, iucA encodes a key enzyme; its deletion abolishes aerobactin production (Bailey et al ., 2018). And iucA deletion mutants exhibit markedly attenuated survival in human ascites and serum, along with reduced virulence in murine pneumonia models (Lee et al ., 2016; Pan et al ., 2008). Caco-2 cells, a widely used in vitro model of intestinal epithelium, were employed in this study due to their ability to form well-differentiated apical and basolateral surfaces with intact tight junction structures during prolonged culture(Finlay & Falkow, 1990). Using this model system, we examined the infectivity of both wild-type hvKP and its isogenic iucA deletion mutant in intestinal epithelial cells. Furthermore, we elucidated the underlying molecular mechanisms through interaction transcriptome analysis. Materials and methods 1.1 Bacterial strains and cell culture The hvKP wild-type strain KPN234 (K20 capsular serotype) was clinically isolated from a patient at Guangdong Nongken Central Hospital. Whole-genome sequencing revealed that this strain carries five classical virulence genes ( iroB , iucA , rmpA2 , rmpA , and peg344 ) on its plasmid. The complete genome sequence has been deposited in the NCBI database (GenBank accession number: GCA_017815715.1). Using suicide plasmid-mediated homologous recombination as previously described by Zi et al. ( 2023 ), we generated isogenic deletion mutants of KPN234, specifically KPN234 ΔiucA , for subsequent functional studies. Caco-2 cells were purchased from Guangzhou Xinyuan technology Co., Ltd. (China). The cells were cultured in high-glucose DMEM (Dulbecco's Modified Eagle Medium; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; heat-inactivated at 56°C for 30 minutes) and 1% non-essential amino acids (Gibco, USA). Cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . 1.2 Siderophore secretion assay Overnight bacterial suspensions were diluted 1:100 in MM9 liquid medium and incubated for 48 hours. The turbidity of each suspension was adjusted to 5 McF, and 1 mL aliquots were transferred to 1.5 mL microcentrifuge tubes. Following centrifugation at 8, 000 × g for 20 minutes at 4°C, supernatants were collected for analysis. For the chromazurol S (CAS) assay, 100 µL of each supernatant or siderophore standard was aliquoted into a 96-well plate, followed by sequential addition of 100 µL CAS solution and 1 µL shuttle solution. After thorough mixing, reactions were incubated at room temperature for 30 minutes, with MM9 medium serving as the negative control. Absorbance at 630 nm was measured using a microplate reader. A standard curve was generated by plotting the OD630 values (Y-axis) against known siderophore concentrations (X-axis), which enabled quantification of siderophore secretion levels in both hvKP-WT and hvKP ΔiucA strains. 1.3 Growth curve analysis Overnight cultures of bacterial single colonies grown on LB agar plates were inoculated into 3 mL of LB liquid medium and incubated at 37°C. When the bacterial density reached an OD600 of approximately 1.2, the cultures were diluted 50-fold with fresh LB medium and transferred to a 96-well plate. The OD600 values were measured at 2-hour intervals using a microplate reader. Growth curves were generated and analyzed using GraphPad Prism software (version 8.0). 1.4 Bacterial adhesion and invasion assays Caco-2 cells were seeded in 24-well plates (5×10⁵ cells/well) and cultured overnight for attachment. Bacterial cultures (LB medium, 37°C, 180 rpm) were diluted 1:100 in fresh LB and grown to mid-log phase (OD600 0.4–0.6). After PBS washing and resuspension, bacteria were adjusted to MOI 100:1. For adhesion assays, cell monolayers were washed, incubated with bacterial suspensions in DMEM (200 × g, 5 min), and maintained at 37°C / 5% CO₂ for 20 min. Following HBSS washes, cells were lysed (0.2% Triton X-100) and lysates pour-plated on LB agar for CFU enumeration (37°C, overnight). Invasion assays followed similar initial steps, with incubation for 2 or 4 h. After HBSS washes, cells were treated with gentamicin (100 µg/ml) in high-glucose DMEM/10% FBS for 1.5 h to eliminate extracellular bacteria prior to lysis and plating. 1.5 Bacterial translocation assay Caco-2 cells were seeded in the apical chamber of 24-well transwell plates (6.5 mm diameter, 3.0 µm pore size; Corning, USA) at 5×10⁵ cells/well (200 µL medium). The basolateral chamber contained 600 µL medium. After 13 days of culture when transepithelial electrical resistance (TEER) exceeded 300 Ω·cm² (measured using RE1600 voltohmmeter; KingTech, Beijing), the cells formed tight monolayers resembling intestinal mucosal epithelium (intestinal mucosal organoids, IMO) ) (Baker & Graham, 2010 ). For infection, approximately 2×10⁶ bacteria were added to each apical chamber and cultured at 37 ℃ with 5% CO₂. TEER was measured at 0 h, 2 h, and 4 h post-infection. Culture medium from basolateral chambers was collected and pour-plated on LB agar for CFU enumeration (3 wells per time point). 1.6 Interaction transcriptomics assay Caco-2 cells were grown in the apical chamber of 24-well transwell plates to form a tight monolayer, as described above. Five experimental groups were established (n = 3 per group): Caco-2 infected with KPN234, Caco-2 infected with KPN234 ΔiucA , Uninfected Caco-2 control (PBS-treated), KPN234 pure culture, and KPN234 ΔiucA pure culture. For infection groups, approximately 2×10⁶ CFU bacteria were added to the apical chamber. Pure culture controls were prepared by inoculating equivalent bacterial numbers into cell culture medium (high-glucose DMEM supplemented with 10% heat-inactivated FBS and 1% non-essential amino acids). All cultures were maintained at 37 ℃ / 5% CO₂ for 4 hours. Following incubation, apical chambers of 24-well transwell plates in three Caco-2 groups were washed with PBS three times, then membranes were excised and flash-frozen in liquid nitrogen immediately. Bacteria in pure culture groups were pelleted by centrifugation (12,000 × g, 1 min) and flash-frozen in liquid nitrogen. Total RNA of each sample was extracted, and rRNA-depleted libraries were prepared using Ribo-off rRNA Depletion Kit (Vazyme, Cat. NO. N406 for human, Cat. No. N407 for bacteria) and KC-DigitalTM Stranded mRNA Library Prep Kit for Illumina® (Catalog NO. DR08502, Wuhan Seqhealth Co., Ltd. China), which adds a unique molecular identifier (UID) consisting of 12 random bases to the cDNA molecules prior to amplification in order to correct for the preferences and errors generated during the PCR and sequencing process. Libraries (200–500 bp) were quantified and sequenced on the DNBSEQ-T7 platform (MGI Tech Co., Ltd. China) using PE150 mode. Raw sequencing data were processed using fastp (v0.23.0) for quality control, followed by UMI-based clustering (95% similarity threshold) to generate consensus sequences. Cleaned reads were aligned to the Homo sapiens (GRCh38; GCA_000001405.29) and K. pneumoniae (GCA_017815715.1) genomes using STAR (v2.5.3a) with default parameters. Read counts mapped to exon regions were quantified using featureCounts (Subread-1.5.1, Bioconductor platform) with RPKM normalization. Differential expression analysis was performed using edgeR (v3.28.1) with thresholds of |log2FC|≥1 (2-fold change) and FDR < 0.05. Significant differentially expressed genes were subjected to KEGG pathway enrichment analysis using KOBAS (v2.1.1; P < 0.05), with visualization in R (v4.4.0). The raw sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1330555. Results 2.1 Siderophore gene iucA deletion resulted in a highly significant decrease in hvKP siderophore secretion The iucA deletion resulted in a significant reduction in siderophore secretion by hvKP, with the ΔiucA mutant (KPN234ΔiucA) producing only 24.0% of the wild-type (KPN234) levels ( Fig. 1a ). Notably, the iucA deletion did not significantly affect bacterial growth curve in LB liquid medium ( Fig. 1b ). 2.2 Deletion of the iucA gene impairs hvKP infectivity in Caco-2 cells The invasion/adhesion ratio of wild-type KPN234 was significantly higher than that of the ΔiucA mutant at both 2h and 4h post-infection ( p < 0.05). While KPN234 showed time-dependent enhancement of invasion (2h vs 4h, p < 0.05), the ΔiucA strain exhibited no significant change ( Fig. 2a ). Transwell assays revealed that only KPN234 could traverse the Caco-2 monolayer after 4h, with no detectable translocation observed for the ΔiucA mutant ( Fig. 2b ). 2.3 Wild-type and ΔiucA hvKP do not disrupt tight junction integrity in Caco-2 monolayers TEER measurements demonstrated no significant differences in transepithelial resistance between KPN234-infected, KPN234 ΔiucA -infected, and PBS control groups at any time point ( Fig. 3 ). In contrast, EGTA-treated positive controls showed rapid TEER reduction (>80% decrease by 2h post-treatment), approaching baseline levels observed in cell-free inserts. These results indicate that hvKP translocation across Caco-2 monolayers within 4h of infection occurs without compromising tight junction integrity. 2.4 IucA knockout alters the transcriptional profile of hvKP during infection of Caco-2 monolayers To investigate host-pathogen interactions, we performed dual RNA-seq analysis of hvKP and Caco-2 monolayers at 4 h post-infection. Principal component analysis revealed: after exposure to Caco-2 monolayers, the intra-group variability in all sets increased markedly, reflected by a pronounced separation between individual samples. Moreover, the cell-exposed and medium-only groups were clearly segregated, indicating that hvKP underwent substantial transcriptional reprogramming upon interaction with the Caco-2 monolayer ( Fig. 4a ). Compared with the control group cultured in medium alone, wild-type hvKP exhibited significant differential expression of 1,446 genes following interaction with the Caco-2 monolayer ( Table S1 ). Among them, 15 KEGG pathways were enriched for up-regulated genes, whereas 53 pathways were enriched for down-regulated genes ( Table S2, S3 ). The five pathways with the highest enrichment scores that were exclusively up-regulated were Ribosome, Protein export (SecG, SecM, tatA, tatB), Bacterial secretion system (T2SS: GspK, GspL; T6SS: tssD), Fatty acid biosynthesis, and RNA polymerase ( Fig. 4b-d ). Conversely, the five most highly enriched pathways showing exclusive down-regulation were C5-Branched dibasic acid metabolism, Lipopolysaccharide biosynthesis, Synthesis and degradation of ketone bodies, Biosynthesis of siderophore group nonribosomal peptides, and Biotin metabolism ( Fig. 4b, c ). Following infection of Caco-2 monolayers, the ΔiucA mutant exhibited significant downregulation of ribosome biogenesis, ABC transporters, and two-component system pathways compared to wild-type hvKP ( Table S4; Fig. 4e ). These transcriptional alterations correlate with the observed impaired proliferative capacity of the ΔiucA mutant in Caco-2 cells. Concurrently, the mutant upregulated multiple metabolic pathways when exposed to Caco-2 monolayers, including branched-chain amino acid biosynthesis, biotin/CoA production, and glutathione metabolism, demonstrating compensatory metabolic rewiring to enhance nutrient acquisition, redox balance, and environmental adaptation following iucA loss ( Table S5; Fig. 4d ). 2.5 Impact of iucA deletion on the global transcriptomic response of Caco-2 monolayers to hvKP infection PCA showed that both wild-type and ΔiucA hvKP groups separated clearly from PBS controls, confirming that infection provokes a profound transcriptional reprogramming of the epithelial layer ( Fig. 5a ). Compared with PBS, Caco-2 monolayers exposed to wild-type hvKP KPN234 displayed 1,851 significant DEGs, with 12 up-regulated KEGG pathways and 21 down-regulated pathways ( Table S6-8; Fig. 5b ). The most enriched up-regulated pathway was pentose and glucuronate interconversions, followed by peroxisome, while the TGF-β signaling pathway was also significantly induced ( Fig. 5c ). Among repressed pathways, protein digestion and absorption showed the strongest enrichment, accompanied by marked down-regulation of oxidative phosphorylation ( Fig. 5d ). Concurrent upregulation of T2SS (gspK/gspL) and T6SS (tssD) genes in hvKP, coupled with host protein metabolism pathway suppression, implies potential disruption of Caco-2 proteostasis by bacterial effectors (Zhou, et al ., 2022). Finally, the host autophagy pathway was significantly suppressed in infected monolayers ( Fig. 5d, e ). However, the two bacterial groups clustered together, and DEG analysis revealed only nine genes with |log2FC| > 1 (max = 1.45), indicating that loss of iucA does not markedly alter host transcriptional signatures ( Table S9 ). Discussions Siderophores are critical virulence factors for hvKP, and deletion of siderophore biosynthesis genes leads to a significant reduction in hvKP virulence (Siu et al., 2011 ). hvKP produces markedly higher levels of siderophores than cKP, particularly in iron-depleted environments such as human body fluids. However, the role of siderophores in hvKP’s breaching of the intestinal mucosal barrier remains incompletely understood. Our results demonstrate that iucA knockout reduces total siderophore secretion by nearly 80% in basal bacterial culture media. Russo et al. ( 2015 ) reported even more pronounced reductions—95% in ascites, 94% in urine, and 100% in serum—when iucA was mutated, suggesting that nutrient-rich media may partially mask the impact of siderophore deficiency. Hsu et al. (2014) showed that systemic infection-causing KP strains of the K2 capsular serotype can adhere to, invade, and translocate across Caco-2 cell monolayers. Our study reveals that the K20 serotype hvKP strain KPN234 (wild-type) shares these capabilities, though with comparatively weaker adhesion/invasion efficiency (< 0.6% vs. 1.46% ± 0.18% reported by Hsu et al. ). This discrepancy may stem from KPN234’s hypermucoviscous phenotype, as studies suggest mucoidity negatively correlates with bacterial invasiveness (Xu, et al., 2021 ). Transepithelial electrical resistance (TEER) assays confirmed that hvKP traverses Caco-2 monolayers without disrupting tight junctions, consistent with findings of Hsu et al. (2014). However, at the transcriptional level, we still observed significant downregulation of tight junction pathways in Caco-2 cell monolayers following hvKP infection (Table S8 ), suggesting that prolonged interaction with hvKP may ultimately compromise epithelial barrier integrity. Although iucA deletion did not impair hvKP’s in vitro proliferation, it significantly attenuated adhesion, invasion, and monolayer translocation. Parallel observations were reported by Deng et al. (2022): iucA deficiency in uropathogenic E. coli (UPEC) reduced adhesion to bladder epithelial cells, impaired bladder colonization in mice, and hindered urothelial transmigration. Notably, iucA knockout also downregulated quorum sensing (QS) pathway (including LuxS, QseC, and 12 other genes) in infected Caco-2 monolayers—a system tightly linked to biofilm formation (Table S10 ). Prior work has reported that siderophore production positively regulates biofilm development (Sadiq et al ., 2024). While iucA knockout caused an 80% decline in siderophore secretion (MM9 medium), it did not alter the transcriptional profile of Caco-2 monolayers. Thus, iucA ’s influence on infectivity likely stems from its regulation of bacterial physiology, including metabolic and signaling adaptations. This contrasts with in vivo findings: Wu et al . demonstrated that iucA -deficient hvKP induced less hepatic oxidative stress and tissue damage in mice than the wild-type (Wu, et al., 2022 ). Siderophores typically affect host cells indirectly—e.g., by sequestering iron to disrupt host oxidative burst capacity (Marchetti, et al., 2020 ) or modulating immune responses via other virulence factors (Sheldon, et al., 2015 ). The divergence between our in vitro and published in vivo results may reflect fundamental differences in iron dynamics: systemic immune responses (e.g., neutrophil recruitment or macrophage polarization) likely amplify siderophore-mediated pathology in vivo, whereas monoculture models lack such multicellular crosstalk. Future in vivo intestinal infection models should elucidate iucA 's mechanistic role in hvKP gut translocation. Transcriptomic analysis revealed that hvKP infection significantly activates the pentose and glucuronate interconversions pathway in Caco-2 cell monolayers. This metabolic pathway converts pentoses into glucuronate, which subsequently enters the pentose phosphate pathway (PPP) to facilitate NADPH generation (Vo, et al., 2021 ), suggesting that hvKP may reprogram the host metabolic network to meet its energy and metabolic precursor demands. Notably, multiple pathogens including Cryptococcus neoformans , Chlamydia , and Plasmodium species have been shown to hijack the host PPP to acquire essential metabolites (Oyelade, et al., 2019 ; Price, et al., 2011 ; Yang, et al., 2024 ).. The specific upregulation of this pathway in Caco-2 cells observed in our study likely represents a host metabolic adaptive response to hvKP infection, while also implying that hvKP may similarly exploit host metabolic resources to sustain its infectious progression. We also detected significant upregulation of the TGF-β signaling pathway in infected cell monolayers. As a well-characterized immunosuppressive pathway, TGF-β activation typically facilitates pathogen survival during infection (Deng et al., 2024 ), which is consistent with previous reports of its induction during classical KP (cKP) infection (Liu et al ., 2022). Surprisingly, our data revealed marked downregulation of autophagy pathways in hvKP-infected Caco-2 monolayers. This finding appears paradoxical because: (1) impaired autophagy has been shown to increase intestinal barrier permeability (Wu et al., 2015 ; Yang et al., 2018 ); and (2) most enteric pathogens (e.g., Salmonella, Enterococcus, Shigella) actually activate epithelial autophagy as part of host defense mechanisms et al ., 2019). While KP infection has been reported to induce autophagy in macrophages and alveolar epithelial cells (Shi et al., 2023 ; Wang et al., 2023 ), our results indicate that hvKP may employ a distinct strategy by suppressing autophagy specifically in intestinal epithelial cells. Considering the reported tissue-specific adaptation of KP virulence mechanisms in a previous study (Holmes et al., 2023 ), the observed autophagy inhibition in gut epithelium may suggest a unique host–pathogen interaction that merits further investigation. Declarations Acknowledgements This research was financially supported by Hundred Young Researchers Grant Program [grant number GDMUD2023004] and Dongguan Science and Technology of Social Development Program [grant number 20231800936202]. Author contribution Tingting Zhi designed the experiments, wrote the original draft, and acquired funding . Chunjie Zhu designed the experiments, analyzed the data, and acquired funding . Zi Caiqin performed the experiments and analyzed the data. Jinxiu Xie analyzed the data and visualization of the data . Ruijun Lao performed the experiments. Liping Wang provided expert technical guidance on cell culture methodologies . Jiangmei Gao provided expert advice on data interpretation . Na Mi conceived the study andsupervised the research. Zuguo Zhao supervised the research, reviewed and edited the manuscript. Data availability The interaction transcriptomics datasets of this article are available in the NCBI SRA under BioProject accession number PRJNA1330555 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1330555). Conflict of interest The authors declare no competing interests. References Bailey, D. C., Alexander, E., Rice, M. R., Drake, E. J., Mydy, L. S., Aldrich, C. C., & Gulick, A. M. (2018). Structural and functional delineation of aerobactin biosynthesis in hypervirulent Klebsiella pneumoniae . J Biol Chem , 293 (20), 7841-52. doi: 10.1074/jbc.RA118.002798 Baker, N. T., & Graham, L. L. (2010). Campylobacter fetus translocation across Caco-2 cell monolayers. Microb Pathog, 49 (5), 260-72. doi: 10.1016/j.micpath.2010.06.008 Choby, J. E., Howard-Anderson, J., & Weiss, D. S. (2020). Hypervirulent Klebsiella pneumoniae - clinical and molecular perspectives. J Intern Med, 287 (3), 283-300. doi: 10.1111/joim.13007 Deng, C., Kang, J. L., Lin, M. S., & Lin, Y. F.(2022). The effect of iucA gene deletion on the proliferation, adhesion, invasion and colonization ability of Uropathogenic E. coli . Acta Univ Med Anhui , 57 (02), 177-81. doi: 10.19405/j.cnki.issn1000-1492.2022.02.003. Deng, Z., Fan, T., Xiao, C., Tian, H., Zheng, Y., Li, C., & He, J. (2024). TGF-β signaling in health, disease and therapeutics. Signal Transduct Target Ther , 9 (1), 61. doi: 10.1038/s41392-024-01764-w Finlay, B. B., & Falkow, S. (1990). Salmonella interactions with polarized human intestinal Caco-2 epithelial cells. J Infect Dis, 162 (5), 1096-106. doi: 10.1093/infdis/162.5.1096 Fung, C. P., Lin, Y. T., Lin, J. C., Chen, T. L., Yeh, K. M., Chang, F. Y., Chuang, H. C., Wu, H. S., Tseng, C. P., & Siu, L. K. (2012). Klebsiella pneumoniae in gastrointestinal tract and pyogenic liver abscess. Emerg Infect Dis, 18 (8), 1322-5. doi: 10.3201/eid1808.111053 Haq, S., Grondin, J., Banskota, S., & Khan, W. I. (2019). Autophagy: roles in intestinal mucosal homeostasis and inflammation. J Biomed Sci, 26 (1), 19. doi: 10.1186/s12929-019-0512-2 Holmes, C. L., Wilcox, A. E., Forsyth, V., Smith, S. N., Moricz, B. S., Unverdorben, L. V., Mason, S., Wu, W., Zhao, L., Mobley, H. L. T., & Bachman, M. A. (2023). Klebsiella pneumoniae causes bacteremia using factors that mediate tissue-specific fitness and resistance to oxidative stress. PLoS Pathog, 19 (7), e1011233. doi: 10.1371/journal.ppat.1011233 Hsu, C. R., Pan, Y. J., Liu, J. Y., Chen, C. T., Lin, T. L., & Wang, J. T. (2015). Klebsiella pneumoniae translocates across the intestinal epithelium via Rho GTPase- and phosphatidylinositol 3-kinase/Akt-dependent cell invasion. Infect Immun, 83 (2), 769-79. doi: 10.1128/iai.02345-14 Kocsis, B. (2023). Hypervirulent Klebsiella pneumoniae : An update on epidemiology, detection and antibiotic resistance. Acta Microbiol Immunol Hung , 70 (4), 278-87. doi: 10.1556/030.2023.02186 Lan, P., Shi, Q., Zhang, P., Chen, Y., Yan, R., Hua, X., Jiang, Y., Zhou, J., & Yu, Y. (2020). Core Genome Allelic Profiles of Clinical Klebsiella pneumoniae Strains Using a Random Forest Algorithm Based on Multilocus Sequence Typing Scheme for Hypervirulence Analysis. J Infect Dis, 221 (Suppl 2), S263-71. doi: 10.1093/infdis/jiz562 Lee, C. R., Lee, J. H., Park, K. S., Jeon, J. H., Kim, Y. B., Cha, C. J., Jeong, B. C., & Lee, S. H. (2017). Antimicrobial Resistance of Hypervirulent Klebsiella pneumoniae : Epidemiology, Hypervirulence-Associated Determinants, and Resistance Mechanisms. Front Cell Infect Microbiol., 7, 483. doi: 10.3389/fcimb.2017.00483 Lee, I. R., Molton, J. S., Wyres, K. L., Gorrie, C., Wong, J., Hoh, C. H., Teo, J., Kalimuddin, S., Lye, D. C., Archuleta, S., Holt, K. E., & Gan, Y. H. (2016). Differential host susceptibility and bacterial virulence factors driving Klebsiella liver abscess in an ethnically diverse population. Sci Rep, 6 , 29316. doi: 10.1038/srep29316 Liu, C., & Guo, J. (2019). Hypervirulent Klebsiella pneumoniae (hypermucoviscous and aerobactin positive) infection over 6 years in the elderly in China: antimicrobial resistance patterns, molecular epidemiology and risk factor. Ann Clin Microbiol Antimicrob, 18 (1), 4. doi: 10.1186/s12941-018-0302-9 Marchetti, M., De Bei, O., Bettati, S., Campanini, B., Kovachka, S., Gianquinto, E., Spyrakis, F., & Ronda, L. (2020). Iron Metabolism at the Interface between Host and Pathogen: From Nutritional Immunity to Antibacterial Development. Int J Mol Sci, 21 (6), 2415. doi: 10.3390/ijms21062145 Nguyen, Q., Nguyen, Y. T. P., Ha, T. T., Tran, D. T. N., Voong, P. V., Chau, V., Nguyen, P. L. N., Le, N. T. Q., Nguyen, L. P. H., Nguyen, T. T. N., Trinh, T. V., Carrique-Mas J. J., Baker, S., Thwaites, G., Rabaa, M. A., Choisy, M., Chung, H. T., & Pham, D. T. (2024). Genomic insights unveil the plasmid transfer mechanism and epidemiology of hypervirulent Klebsiella pneumoniae in Vietnam. Nat Commun, 15 (1), 4187. doi: 10.1038/s41467-024-48206-3 Oyelade, J., Isewon, I., Aromolaran, O., Uwoghiren, E., Dokunmu, T., Rotimi, S., Aworunse, O., Obembe, O., & Adebiyi, E. (2019). Computational Identification of Metabolic Pathways of Plasmodium falciparum using thek-Shortest Path Algorithm. Int J Genomics, 2019 , 1-13. doi: 10.1155/2019/1750291 Pan, Y. J., Fang, H. C., Yang, H. C., Lin, T. L., Hsieh, P. F., Tsai, F. C., Keynan, Y., & Wang, J. T. (2008). Capsular polysaccharide synthesis regions in Klebsiella pneumoniae serotype K57 and a new capsular serotype. J Clin Microbiol, 46 (7), 2231-40. doi: 10.1128/jcm.01716-07 Price, M. S., Betancourt-Quiroz, M., Price, J. L., Toffaletti, D. L., Vora, H., Hu, G., Kronstad, J. W., Perfect, J. R., & Dromer, F. (2011). Cryptococcus neoformans Requires a Functional Glycolytic Pathway for Disease but Not Persistence in the Host. mBio, 2 (3), e00103-11. doi: 10.1128/mBio.00103-11 Russo, T. A., Olson, R., MacDonald, U., Beanan, J., & Davidson, B. A. (2015). Aerobactin, but not yersiniabactin, salmochelin, or enterobactin, enables the growth/survival of hypervirulent (hypermucoviscous) Klebsiella pneumoniae ex vivo and in vivo. Infect Immun, 83 (8), 3325-33. doi: 10.1128/iai.00430-15 Russo, T. A., Olson, R., Macdonald, U., Metzger, D., Maltese, L. M., Drake, E. J., & Gulick, A. M. (2014). Aerobactin mediates virulence and accounts for increased siderophore production under iron-limiting conditions by hypervirulent (hypermucoviscous) Klebsiella pneumoniae . Infect Immun, 82 (6), 2356-67. doi: 10.1128/iai.01667-13 Sadiq Aniz, E. H., & Jassim, Y. A. (2024). Anticancer activity of pyoverdine (PVD) producing by antibiotic-resistant Pseudomonas aeruginosa isolated from burn and wound infections. J Appl Nat Sci, 16 (2). doi: 10.31018/jans.v16i2.5506 Sheldon, J. R., Heinrichs, D. E., & Whitfield, C. (2015). Recent developments in understanding the iron acquisition strategies of gram positive pathogens. FEMS Microbiol Rev, 39 (4), 592-630. doi: 10.1093/femsre/fuv009 Shi, Z. Y., Yin, X., Hou, X., Wang, L. Q., Li, G., Wang, J., Wang, W., Zhang, Y., Li, S., & Jia, W. (2023). Mechanism of Notch Signal Regulating Alveolar Epithelial Cell Autophagy after Infection with Klebsiella Pneumoniae . Clin Lab, 69 (5). doi: 10.7754/Clin.Lab.2022.211203 Siu, L. K., Fung, C. P., Chang, F. Y., Lee, N., Yeh, K. M., Koh, T. H., & Ip, M. (2011). Molecular typing and virulence analysis of serotype K1 Klebsiella pneumoniae strains isolated from liver abscess patients and stool samples from noninfectious subjects in Hong Kong, Singapore, and Taiwan. J Clin Microbiol, 49 (11), 3761-5. doi: 10.1128/jcm.00977-11 Spellberg, B., Russo, T. A., Shon, A. S., Beanan, J. M., Olson, R., MacDonald, U., Pomakov, A. O., & Visitacion, M. P. (2011). Hypervirulent K. Pneumoniae Secretes More and More Active Iron-Acquisition Molecules than “Classical” K. Pneumoniae Thereby Enhancing its Virulence. PLoS ONE, 6 (10), e26734. doi: 10.1371/journal.pone.0026734 Vo, P. N. L., Lee, H. M., Ren, J., & Na, D. (2021). Optimized expression of Hfq protein increases Escherichia coli growth. J Biol Eng, 15 (1). doi: 10.1186/s13036-021-00260-x Wang, X., Bi, C., Xin, X., Zhang, M., Fu, H., Lan, L., Wang, M., & Yan, Z. (2023). Pyroptosis, apoptosis, and autophagy are involved in infection induced by two clinical Klebsiella pneumoniae isolates with different virulence. Front Cell Infect Microbiol, 13 , 1165609. doi: 10.3389/fcimb.2023.1165609 Wu, J., Chen, J., Wang, Y., Meng, Q., & Zhao, J. (2022). Siderophore iucA of hypermucoviscous Klebsiella pneumoniae promotes liver damage in mice by inducing oxidative stress. Biochem Biophys Rep, 32 , 101376. doi: 10.1016/j.bbrep.2022.101376 Wu, S., Zhang, Y. G., Lu, R., Xia, Y., Zhou, D., Petrof, E. O., Claud, E. C., Chen, D., Chang, E. B., Carmeliet, G., & Sun, J. (2015). Intestinal epithelial vitamin D receptor deletion leads to defective autophagy in colitis. Gut, 64 (7), 1082-94. doi: 10.1136/gutjnl-2014-307436 Xu, Q., Yang, X., Chan, E. W. C., & Chen, S. (2021). The hypermucoviscosity of hypervirulent K. pneumoniae confers the ability to evade neutrophil-mediated phagocytosis. Virulence, 12 (1), 2050-9. doi: 10.1080/21505594.2021.1960101 Yang, L., Liu, C., Zhao, W., He, C., Ding, J., Dai, R., Xu, K., Xiao, L., Luo, L., Liu, S., Li, W., & Meng, H. (2018). Impaired Autophagy in Intestinal Epithelial Cells Alters Gut Microbiota and Host Immune Responses. Appl Environ Microbiol, 84 (18), e00880-18. doi: 10.1128/aem.00880-18 Yang, S., Zeng, J., Yu, J., Sun, R., Tuo, Y., & Bai, H. (2024). Insights into Chlamydia Development and Host Cells Response. Microorganisms, 12 (7), 1302. doi: 10.3390/microorganisms12071302 Zhou, H., Beltrán, J. F., & Brito, I. L. (2022). Host-microbiome protein-protein interactions capture disease-relevant pathways. Genome Biol, 23 (1), 72. doi: 10.1186/s13059-022-02643-9 Zi, C., Yang, S., Fu, X. F., Wang, W. Q., Luo, Y., Zhang, J., Guo, W. T., Wang, H. P., Li, L., Liang, X. Y., Mi, N., Zhi, T. T, & Zhao, Z. G. (2023). An efficient method for knocking out genes on the virulence plasmid of hypervirulent Klebsiella pneumoniae . New Microbiol, 46 (2), 186-95. Additional Declarations No competing interests reported. Supplementary Files TableS1.DifferentiallyexpressedgenesinWTKPN234afterexposuretoCaco2monolayerscomparedtopurecultureincellculturemedium.xls TableS2.KEGGpathwaysupregulatedinWTKPN234followingexposuretoCaco2monolayersversuspurecultureincellculturemedium.xls TableS3.KEGGpathwaysdownregulatedinWTKPN234followingexposuretoCaco2monolayersversuspurecultureincellculturemedium.xls TableS4.KEGGpathwaysdownregulatedinKPN234iucAversusWTKPN234followingexposuretoCaco2monolayers.xls TableS5.KEGGpathwaysupregulatedinKPN234iucAversusWTKPN234followingexposuretoCaco2monolayers.xls TableS6.DifferentiallyexpressedgenesinCaco2monolayersuponexposuretoWTKPN234versusPBScontrols.xls TableS7.KEGGpathwaysupregulatedinCaco2monolayersuponexposuretoWTKPN234versusPBScontrols.xls TableS8.KEGGpathwaysdownregulatedinCaco2monolayersuponexposuretoWTKPN234versusPBScontrols.xls TableS9.DifferentiallyexpressedgenesinCaco2monolayersuponexposuretoKPN234iucAversusWTKPN234.xls TableS10.KEGGpathwaysdownregulatedinKPN234iucAfollowingexposuretoCaco2monolayersversuspurecultureincellculturemedium.xls Cite Share Download PDF Status: Posted 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-7689359","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":525972573,"identity":"66da3f74-5e36-433d-946b-ef875721fb4e","order_by":0,"name":"Tingting Zhi","email":"","orcid":"","institution":"Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Zhi","suffix":""},{"id":525972575,"identity":"624ca64f-ee38-4653-a205-20beaeb40148","order_by":1,"name":"Chunjie Zhu","email":"","orcid":"","institution":"Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chunjie","middleName":"","lastName":"Zhu","suffix":""},{"id":525972578,"identity":"6fa8560d-5d4d-4e94-8a7b-816166edb199","order_by":2,"name":"Caiqin Zi","email":"","orcid":"","institution":"Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Caiqin","middleName":"","lastName":"Zi","suffix":""},{"id":525972579,"identity":"c8fda4d7-a3fc-4a3e-9350-7cb0e4115f5b","order_by":3,"name":"Jinxiu Xie","email":"","orcid":"","institution":"Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinxiu","middleName":"","lastName":"Xie","suffix":""},{"id":525972582,"identity":"93fc9b38-8b12-4aad-831a-deb05b19ec90","order_by":4,"name":"Ruijun Lao","email":"","orcid":"","institution":"Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ruijun","middleName":"","lastName":"Lao","suffix":""},{"id":525972586,"identity":"ab6f99ee-02f7-4acd-a2da-be8eb6574985","order_by":5,"name":"Liping Wang","email":"","orcid":"","institution":"Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liping","middleName":"","lastName":"Wang","suffix":""},{"id":525972589,"identity":"9b244c4e-4eb5-42c4-ab2e-c3d488adccb1","order_by":6,"name":"Jiangmei Gao","email":"","orcid":"","institution":"Guangdong Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jiangmei","middleName":"","lastName":"Gao","suffix":""},{"id":525972590,"identity":"5980179d-7b15-4eb3-9349-d36dd7fc4672","order_by":7,"name":"Na Mi","email":"","orcid":"","institution":"Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Mi","suffix":""},{"id":525972591,"identity":"2dd661b3-3d26-49b3-8e9a-d4eb69017e82","order_by":8,"name":"Zuguo Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYDACCRiDvQHKPkC0Fp4DJGuRSCBSi/zs5ocPf7bVJvZLPr9462YbgxzfjQTGzwV4tDDOOWZsINl2PHHm7Jxi69w2BmPJGwnM0jPwaGGWSDCTMGw7lrjhdk6aNFBL4oYbCWzMPHi0sEmkf5NIBGm5eQaspZ6gFh6JHDOJg201QMPZj4G0JBgQ0iIhkVNs2HDugPHMnhxm65xzEoYzzzxslsanRX5G+saHP8rqZPvZjz+8nVNmI893PPngZ3xaoOAwyI0GDJBoYmwgrIGBoQ6I2R8Qo3IUjIJRMApGIAAAWe9NwDzeKiYAAAAASUVORK5CYII=","orcid":"","institution":"Guangdong Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zuguo","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2025-09-23 04:08:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7689359/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7689359/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93101448,"identity":"8ea45b73-4220-47f4-832e-eb314d97ec14","added_by":"auto","created_at":"2025-10-09 05:19:56","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3473914,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptAVL.doc","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/019360464a61a7d641f3b376.doc"},{"id":93101069,"identity":"1aaf77e2-276b-4e19-a2ce-4363fa1fa94e","added_by":"auto","created_at":"2025-10-09 05:11:56","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9759,"visible":true,"origin":"","legend":"","description":"","filename":"ed502d838ed3498093a658b25f32666f.json","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/0dacf772b98aa1dd5ddc5dbd.json"},{"id":93100342,"identity":"ddabefd3-1b51-46e3-90b0-e74977ad8441","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":374784,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.DifferentiallyexpressedgenesinWTKPN234afterexposuretoCaco2monolayerscomparedtopurecultureincellculturemedium.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/b0bd3a339d8b40c7d2c3dd55.xls"},{"id":93100338,"identity":"94a0e8ce-673e-40ca-9d75-3fe14b577d3a","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"xls","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":18244,"visible":true,"origin":"","legend":"","description":"","filename":"TableS10.KEGGpathwaysdownregulatedinKPN234iucAfollowingexposuretoCaco2monolayersversuspurecultureincellculturemedium.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/054ee71b81dee43f24394ac3.xls"},{"id":93101071,"identity":"0cc53a55-eb05-4f8f-a947-c3b31a7288b8","added_by":"auto","created_at":"2025-10-09 05:11:56","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":31232,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.KEGGpathwaysupregulatedinWTKPN234followingexposuretoCaco2monolayersversuspurecultureincellculturemedium.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/da5ca1055f69e49bd8acfb9e.xls"},{"id":93100348,"identity":"a2edf25f-2c6e-4cd4-b71b-118f5eec9fff","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"xls","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":57344,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.KEGGpathwaysdownregulatedinWTKPN234followingexposuretoCaco2monolayersversuspurecultureincellculturemedium.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/e44d334de0590afeacaf652c.xls"},{"id":93101088,"identity":"f65d609f-4394-41ef-b8e6-3c93fd661737","added_by":"auto","created_at":"2025-10-09 05:11:56","extension":"xls","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":26624,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.KEGGpathwaysdownregulatedinKPN234iucAversusWTKPN234followingexposuretoCaco2monolayers.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/dda8a2ec75f73e5dfb20b414.xls"},{"id":93102009,"identity":"7107a759-bffe-493c-83f6-38c1affd00fd","added_by":"auto","created_at":"2025-10-09 05:27:56","extension":"xls","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8193,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.KEGGpathwaysupregulatedinKPN234iucAversusWTKPN234followingexposuretoCaco2monolayers.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/e99f3cef1278480f27fb8def.xls"},{"id":93101084,"identity":"168667bf-b005-4133-9ba5-36d861ebf07a","added_by":"auto","created_at":"2025-10-09 05:11:56","extension":"xls","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":485888,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6.DifferentiallyexpressedgenesinCaco2monolayersuponexposuretoWTKPN234versusPBScontrols.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/60e30f009df62e095532b185.xls"},{"id":93101079,"identity":"6e83b46d-2cd4-426c-b743-87a043eb7c89","added_by":"auto","created_at":"2025-10-09 05:11:56","extension":"xls","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3345,"visible":true,"origin":"","legend":"","description":"","filename":"TableS7.KEGGpathwaysupregulatedinCaco2monolayersuponexposuretoWTKPN234versusPBScontrols.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/eb8b3f7b1c804226dd21ff3a.xls"},{"id":93101082,"identity":"347b6c3f-25df-430f-9f09-399dc4965553","added_by":"auto","created_at":"2025-10-09 05:11:56","extension":"xls","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9055,"visible":true,"origin":"","legend":"","description":"","filename":"TableS8.KEGGpathwaysdownregulatedinCaco2monolayersuponexposuretoWTKPN234versusPBScontrols.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/b749b83cc54dbf1775296446.xls"},{"id":93100371,"identity":"b0cb5db9-9364-4af7-acde-92afa4ab8699","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"xls","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1909,"visible":true,"origin":"","legend":"","description":"","filename":"TableS9.DifferentiallyexpressedgenesinCaco2monolayersuponexposuretoKPN234iucAversusWTKPN234.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/dbc14875a835df80134e45a1.xls"},{"id":93100362,"identity":"31d61a7e-c25c-4a24-b5a2-71a3f4e5562b","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121775,"visible":true,"origin":"","legend":"","description":"","filename":"ed502d838ed3498093a658b25f32666f1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/1fd6eee5c7a86f06283cda01.xml"},{"id":93100368,"identity":"b76006d6-4252-40e0-94fe-14e5fd215ff9","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":47434,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/8ca34c73f76975d1a946f7ef.png"},{"id":93100366,"identity":"cc55d7c0-9b15-468b-b56c-43ed82e89c77","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":39177,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/660a21d3d227648068f4b022.png"},{"id":93100361,"identity":"c40be02d-eeac-46eb-a567-fcbbba614735","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":44450,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/296f403af9acaa67df444692.png"},{"id":93101087,"identity":"26870465-d661-41d8-bdbd-0339dae595b6","added_by":"auto","created_at":"2025-10-09 05:11:56","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1281042,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/75e75e250e9f1306d19f26e9.png"},{"id":93100373,"identity":"169fe166-a867-4128-b65a-1262876a845d","added_by":"auto","created_at":"2025-10-09 05:03:57","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1361076,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/e6512a3a64040710f5ea75ab.png"},{"id":93100355,"identity":"14964a41-0b48-44b2-b941-9894cb19c8d5","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11971,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/36c2a9ec51811f5fc4cf8d53.png"},{"id":93100367,"identity":"cb132867-3ff6-40fe-9900-610953c9de63","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9410,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/3da9418135e73308869e95d0.png"},{"id":93101085,"identity":"a391feed-6b48-4499-844f-bab897a9870e","added_by":"auto","created_at":"2025-10-09 05:11:56","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8363,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/55bf3a2b3c16aa71b6b895de.png"},{"id":93101089,"identity":"e1ec0ce1-5770-4e3d-a72c-8615b2abff90","added_by":"auto","created_at":"2025-10-09 05:11:56","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161301,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/98e4fdd9e88b4a3e64236653.png"},{"id":93100364,"identity":"220d18f3-a5f2-4791-9964-89f6a8ea5d8e","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161723,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/abf23e93f92fabe6fec1d7bd.png"},{"id":93101090,"identity":"01d595e4-27e7-49db-a600-929a497ea74c","added_by":"auto","created_at":"2025-10-09 05:11:56","extension":"xml","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":119589,"visible":true,"origin":"","legend":"","description":"","filename":"ed502d838ed3498093a658b25f32666f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/6fb47dc597247af72051ba36.xml"},{"id":93100370,"identity":"a25c5870-866d-4013-9d61-84bfd61c51ed","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":134232,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/57b65ee3ce5cd51d4fecb2a8.html"},{"id":93100334,"identity":"4109327a-8be3-403e-bfcf-eb6ac9ba690a","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of iucA deletion on siderophore production and growth in hvKP.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003eSiderophore secretion levels in MM9 medium (48 h). \u003cstrong\u003e(b) \u003c/strong\u003eGrowth curves in LB medium. Data shown as mean ± SD (n=3 biological replicates).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/3dbdb5abf96e93d16910cdc0.png"},{"id":93101441,"identity":"07565acf-0a9e-4f62-826f-d28237a3a267","added_by":"auto","created_at":"2025-10-09 05:19:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eiucA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edeletion on hvKP infectivity in Caco-2 cells. (a)\u003c/strong\u003e Invasion efficiency of hvKP strains (wild-type KPN234 vs. \u003cem\u003eΔiucA\u003c/em\u003e mutant) after 2 h and 4 h infection. Caco-2 monolayers in 24-well plates were infected at MOI 100, and intracellular bacteria were quantified relative to adherent bacteria (mean ± SD; \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 by \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003e(b)\u003c/strong\u003e Transmigration capacity across polarized Caco-2 monolayers. Bacteria penetrating the transwell membrane after 4 h infection were enumerated (median with range; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 by Mann-Whitney \u003cem\u003eU\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/c244ba8607b5dbf8bb41b4ac.png"},{"id":93100337,"identity":"8a797200-0545-41b9-b576-d05e74e92a09","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34517,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of hvKP infection on tight junction integrity in Caco-2 monolayers. \u003c/strong\u003eCaco-2 cells were cultured in Transwell inserts until formation of polarized monolayers (TEER \u0026gt; 300 Ω·cm²), followed by infection with hvKP strains. Transepithelial electrical resistance (TEER) was measured at 0, 2, and 4 h post-infection. EGTA (2 mM), a calcium chelator known to disrupt tight junction proteins, served as positive control. Data represent mean ± SD (n=3).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/c4c779acbc075b3cef7ddc07.png"},{"id":93101073,"identity":"31e33282-8c6c-4e93-9b58-07c350d80906","added_by":"auto","created_at":"2025-10-09 05:11:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":129691,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic reprogramming of hvKP during Caco-2 monolayer infection. (a) \u003c/strong\u003ePrincipal component analysis (PCA) of hvKP strains comparing pure culture in cell culture mediumversus interaction with the Caco-2 monolayer. \u003cstrong\u003e(b) \u003c/strong\u003eHeatmap analysis of differentially expressed genes (DEGs), highlighting the top 15 significantly upregulated and downregulated KEGG pathways in WT-hvKP interacting with Caco-2 monolayersversus pure cultures controls. \u003cstrong\u003e(c)\u003c/strong\u003e Volcano plot of DEGs in wild-type hvKP after 4 h infection of Caco-2 monolayers, with pure culturesas controls. \u003cstrong\u003e(d)\u003c/strong\u003e Top 15 enriched pathways (ranked by enrichment factor) for upregulated genes in Caco-2 monolayers infecting hvKP\u003cem\u003eΔiucA \u003c/em\u003eversus pure cultures controls. \u003cstrong\u003e(e)\u003c/strong\u003eEnriched pathways for downregulated genes in Caco-2 monolayers infecting hvKP\u003cem\u003eΔiucA \u003c/em\u003eversus pure cultures controls. WT in CM: wild-type hvKP cultured in cell culture medium; WT on IMO: wild-type hvKP infected with dense monolayers of Caco-2 cells; \u003cem\u003eΔiucA\u003c/em\u003e in CM: mutant hvKP\u003cem\u003eΔiucA\u003c/em\u003e cultured in cell culture medium; \u003cem\u003eΔiucA\u003c/em\u003e on IMO: mutant hvKPΔiucA infected with dense monolayers of Caco-2 cells.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/a0b0e169609e1f3d1dba52f8.png"},{"id":93102008,"identity":"55f6ae3d-2a7c-40b5-b28a-a00e07cb2161","added_by":"auto","created_at":"2025-10-09 05:27:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":132524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic alterations in Caco-2 monolayers following hvKP infection.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Principal component analysis (PCA) of Caco-2 monolayers pre- and post-hvKP infection. \u003cstrong\u003e(b)\u003c/strong\u003eHeatmap analysis of differentially expressed genes (DEGs), highlighting the top 15 significantly upregulated and downregulated pathways in WT hvKP-infected monolayers versus PBS controls. \u003cstrong\u003e(c)\u003c/strong\u003e Top 12 enriched pathways for upregulated genes in WT hvKP-infected monolayers versus PBS controls (ranked by enrichment factor). \u003cstrong\u003e(d)\u003c/strong\u003e Top 15 enriched pathways for downregulated genes in WT hvKP-infected monolayers versus PBS controls (ranked by enrichment factor). \u003cstrong\u003e(e)\u003c/strong\u003e Volcano plot of DEGs in Caco-2 monolayers 4h post- WT hvKP infection (PBS-treated as control). IMO by WT: \u0026nbsp;Caco-2 monolayers infected with wild-type hvKP; IMO by ∆iucA: Caco-2 monolayers infected with hvKP \u003cem\u003eΔiucA\u003c/em\u003e mutant; CTRL: PBS-treated Caco-2 monolayers.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/a86ec30f6dc1c6406ba0234d.png"},{"id":94057143,"identity":"ce22081e-1758-45b6-a5c4-0fdaaef3ed4d","added_by":"auto","created_at":"2025-10-22 04:46:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1263253,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/414b2d51-88b1-43a9-a79f-79bcd7ae1cb4.pdf"},{"id":93101445,"identity":"9c1ca92c-dabd-4954-8050-42f6ed1996aa","added_by":"auto","created_at":"2025-10-09 05:19:56","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":374784,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.DifferentiallyexpressedgenesinWTKPN234afterexposuretoCaco2monolayerscomparedtopurecultureincellculturemedium.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/1f5c3ff4caa925965492f71a.xls"},{"id":93101442,"identity":"9141f8c0-c015-445d-83ea-24089009c687","added_by":"auto","created_at":"2025-10-09 05:19:56","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31232,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.KEGGpathwaysupregulatedinWTKPN234followingexposuretoCaco2monolayersversuspurecultureincellculturemedium.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/0ddedab2d98a44bceccf428e.xls"},{"id":93101077,"identity":"ef6e194e-a879-4201-85ba-da4f4996fe03","added_by":"auto","created_at":"2025-10-09 05:11:56","extension":"xls","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":57344,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.KEGGpathwaysdownregulatedinWTKPN234followingexposuretoCaco2monolayersversuspurecultureincellculturemedium.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/e429fa9ecc13436c1bf8c1fd.xls"},{"id":93100350,"identity":"d56e1804-1a1f-4f43-b64e-ae0386b723fa","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":26624,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.KEGGpathwaysdownregulatedinKPN234iucAversusWTKPN234followingexposuretoCaco2monolayers.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/2caa51816f992fd9399fdc83.xls"},{"id":93100345,"identity":"f251f0a6-79b4-4eba-828a-6660bd4ef782","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"xls","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":8193,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.KEGGpathwaysupregulatedinKPN234iucAversusWTKPN234followingexposuretoCaco2monolayers.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/ec315d0b5dd66c26650443db.xls"},{"id":93101449,"identity":"0507a7b2-9fa1-4c88-98ed-c7ba6ac905ae","added_by":"auto","created_at":"2025-10-09 05:19:56","extension":"xls","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":485888,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6.DifferentiallyexpressedgenesinCaco2monolayersuponexposuretoWTKPN234versusPBScontrols.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/279d089623a8e1b7497c9784.xls"},{"id":93101447,"identity":"daf4e065-d880-43ba-858a-5d5ac183a846","added_by":"auto","created_at":"2025-10-09 05:19:56","extension":"xls","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":3345,"visible":true,"origin":"","legend":"","description":"","filename":"TableS7.KEGGpathwaysupregulatedinCaco2monolayersuponexposuretoWTKPN234versusPBScontrols.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/582ee55d5024f1eee692c7ab.xls"},{"id":93101444,"identity":"b7cd86f7-ca19-4b65-ba49-05bcd4d72c68","added_by":"auto","created_at":"2025-10-09 05:19:56","extension":"xls","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":9055,"visible":true,"origin":"","legend":"","description":"","filename":"TableS8.KEGGpathwaysdownregulatedinCaco2monolayersuponexposuretoWTKPN234versusPBScontrols.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/39fe44ac7fb710bc0ec18b6c.xls"},{"id":93100353,"identity":"95835e48-d131-4acf-b9f5-04d2c9f4ed0b","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"xls","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":1909,"visible":true,"origin":"","legend":"","description":"","filename":"TableS9.DifferentiallyexpressedgenesinCaco2monolayersuponexposuretoKPN234iucAversusWTKPN234.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/6bc354ec96d9875a63d7069f.xls"},{"id":93100360,"identity":"e2f7b08f-5921-4754-b98b-0ca1f811e63b","added_by":"auto","created_at":"2025-10-09 05:03:56","extension":"xls","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":18244,"visible":true,"origin":"","legend":"","description":"","filename":"TableS10.KEGGpathwaysdownregulatedinKPN234iucAfollowingexposuretoCaco2monolayersversuspurecultureincellculturemedium.xls","url":"https://assets-eu.researchsquare.com/files/rs-7689359/v1/ad10cb8a90d98a1957059eaa.xls"}],"financialInterests":"No competing interests reported.","formattedTitle":"IucA gene contributes to the infectivity of hypervirulent Klebsiella pneumoniae in Caco-2 cell models","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (hvKP) is a highly pathogenic subgroup of \u003cem\u003eK. pneumonia\u003c/em\u003e, possessing a higher virulence than classical \u003cem\u003eK. pneumoniae\u003c/em\u003e (cKP). This strain can cause invasive infections even in healthy individuals and is associated with high mortality rates (4.5%–31%) and severe sequelae (Lan\u0026nbsp;\u003cem\u003eet al\u003c/em\u003e., 2020). Studies indicate that hvKP frequently colonizes the human intestinal mucosa, and its translocation from the gut into the bloodstream serves as a major route for organ and systemic infections (Fung \u003cem\u003eet al\u003c/em\u003e., 2012; Liu \u0026amp; Guo, 2019; Nguyen \u003cem\u003eet al\u003c/em\u003e., 2024). However, the precise molecular mechanisms by which hvKP breaches the intestinal mucosal barrier remain unclear.\u003c/p\u003e\n\u003cp\u003eIron acquisition systems are critical virulence determinants in hvKP, facilitating bacterial proliferation through sequestration of host iron sources. Compared to cKP, hvKP demonstrates significantly enhanced (6- to 10-fold) production of various siderophores, including enterobactin, salmochelin, aerobactin, and yersiniabactin (Kocsis, 2023; Lee \u003cem\u003eet al\u003c/em\u003e., 2017). This elevated siderophore production not only improves hvKP's adaptation to host environments but also enables its survival and replication in iron-restricted niches such as blood and ascites(Spellberg \u003cem\u003eet al\u003c/em\u003e., 2011). Notably, aerobactin - encoded by the \u003cem\u003eiucABCD\u003c/em\u003e gene cluster and constituting over 80% of total hvKP siderophores - emerges as a pivotal virulence factor mediating systemic infection and serves as a molecular hallmark of hvKP strains(Choby, \u003cem\u003eet al\u003c/em\u003e., 2020; Russo \u003cem\u003eet al\u003c/em\u003e., 2014). In the aerobactin biosynthesis pathway of hvKP, \u003cem\u003eiucA\u003c/em\u003e encodes a key enzyme; its deletion abolishes aerobactin production (Bailey \u003cem\u003eet al\u003c/em\u003e., 2018). And \u003cem\u003eiucA\u003c/em\u003e deletion mutants exhibit markedly attenuated survival in human ascites and serum, along with reduced virulence in murine pneumonia models (Lee \u003cem\u003eet al\u003c/em\u003e., 2016; Pan \u003cem\u003eet al\u003c/em\u003e., 2008).\u003c/p\u003e\n\u003cp\u003eCaco-2 cells, a widely used in vitro model of intestinal epithelium, were employed in this study due to their ability to form well-differentiated apical and basolateral surfaces with intact tight junction structures during prolonged culture(Finlay \u0026amp; Falkow, 1990). Using this model system, we examined the infectivity of both wild-type hvKP and its isogenic \u003cem\u003eiucA\u003c/em\u003e deletion mutant in intestinal epithelial cells. Furthermore, we elucidated the underlying molecular mechanisms through interaction transcriptome analysis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e1.1\u003c/b\u003e Bacterial strains and cell culture\u003c/h2\u003e\u003cp\u003eThe hvKP wild-type strain KPN234 (K20 capsular serotype) was clinically isolated from a patient at Guangdong Nongken Central Hospital. Whole-genome sequencing revealed that this strain carries five classical virulence genes (\u003cem\u003eiroB\u003c/em\u003e, \u003cem\u003eiucA\u003c/em\u003e, \u003cem\u003ermpA2\u003c/em\u003e, \u003cem\u003ermpA\u003c/em\u003e, and \u003cem\u003epeg344\u003c/em\u003e) on its plasmid. The complete genome sequence has been deposited in the NCBI database (GenBank accession number: GCA_017815715.1). Using suicide plasmid-mediated homologous recombination as previously described by Zi et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we generated isogenic deletion mutants of KPN234, specifically KPN234\u003cem\u003eΔiucA\u003c/em\u003e, for subsequent functional studies.\u003c/p\u003e\u003cp\u003eCaco-2 cells were purchased from Guangzhou Xinyuan technology Co., Ltd. (China). The cells were cultured in high-glucose DMEM (Dulbecco's Modified Eagle Medium; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; heat-inactivated at 56\u0026deg;C for 30 minutes) and 1% non-essential amino acids (Gibco, USA). Cell cultures were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e1.2\u003c/b\u003e Siderophore secretion assay\u003c/h2\u003e\u003cp\u003eOvernight bacterial suspensions were diluted 1:100 in MM9 liquid medium and incubated for 48 hours. The turbidity of each suspension was adjusted to 5 McF, and 1 mL aliquots were transferred to 1.5 mL microcentrifuge tubes. Following centrifugation at 8, 000 \u0026times; g for 20 minutes at 4\u0026deg;C, supernatants were collected for analysis.\u003c/p\u003e\u003cp\u003eFor the chromazurol S (CAS) assay, 100 \u0026micro;L of each supernatant or siderophore standard was aliquoted into a 96-well plate, followed by sequential addition of 100 \u0026micro;L CAS solution and 1 \u0026micro;L shuttle solution. After thorough mixing, reactions were incubated at room temperature for 30 minutes, with MM9 medium serving as the negative control. Absorbance at 630 nm was measured using a microplate reader.\u003c/p\u003e\u003cp\u003eA standard curve was generated by plotting the OD630 values (Y-axis) against known siderophore concentrations (X-axis), which enabled quantification of siderophore secretion levels in both hvKP-WT and hvKP\u003cem\u003eΔiucA\u003c/em\u003e strains.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e1.3\u003c/b\u003e Growth curve analysis\u003c/h2\u003e\u003cp\u003eOvernight cultures of bacterial single colonies grown on LB agar plates were inoculated into 3 mL of LB liquid medium and incubated at 37\u0026deg;C. When the bacterial density reached an OD600 of approximately 1.2, the cultures were diluted 50-fold with fresh LB medium and transferred to a 96-well plate. The OD600 values were measured at 2-hour intervals using a microplate reader. Growth curves were generated and analyzed using GraphPad Prism software (version 8.0).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e1.4\u003c/b\u003e Bacterial adhesion and invasion assays\u003c/h2\u003e\u003cp\u003eCaco-2 cells were seeded in 24-well plates (5\u0026times;10⁵ cells/well) and cultured overnight for attachment. Bacterial cultures (LB medium, 37\u0026deg;C, 180 rpm) were diluted 1:100 in fresh LB and grown to mid-log phase (OD600 0.4\u0026ndash;0.6). After PBS washing and resuspension, bacteria were adjusted to MOI 100:1.\u003c/p\u003e\u003cp\u003eFor adhesion assays, cell monolayers were washed, incubated with bacterial suspensions in DMEM (200 \u0026times; g, 5 min), and maintained at 37\u0026deg;C / 5% CO₂ for 20 min. Following HBSS washes, cells were lysed (0.2% Triton X-100) and lysates pour-plated on LB agar for CFU enumeration (37\u0026deg;C, overnight). Invasion assays followed similar initial steps, with incubation for 2 or 4 h. After HBSS washes, cells were treated with gentamicin (100 \u0026micro;g/ml) in high-glucose DMEM/10% FBS for 1.5 h to eliminate extracellular bacteria prior to lysis and plating.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e1.5\u003c/b\u003e Bacterial translocation assay\u003c/h2\u003e\u003cp\u003eCaco-2 cells were seeded in the apical chamber of 24-well transwell plates (6.5 mm diameter, 3.0 \u0026micro;m pore size; Corning, USA) at 5\u0026times;10⁵ cells/well (200 \u0026micro;L medium). The basolateral chamber contained 600 \u0026micro;L medium. After 13 days of culture when transepithelial electrical resistance (TEER) exceeded 300 Ω\u0026middot;cm\u0026sup2; (measured using RE1600 voltohmmeter; KingTech, Beijing), the cells formed tight monolayers resembling intestinal mucosal epithelium (intestinal mucosal organoids, IMO) ) (Baker \u0026amp; Graham, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor infection, approximately 2\u0026times;10⁶ bacteria were added to each apical chamber and cultured at 37 ℃ with 5% CO₂. TEER was measured at 0 h, 2 h, and 4 h post-infection. Culture medium from basolateral chambers was collected and pour-plated on LB agar for CFU enumeration (3 wells per time point).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e1.6\u003c/b\u003e Interaction transcriptomics assay\u003c/h2\u003e\u003cp\u003eCaco-2 cells were grown in the apical chamber of 24-well transwell plates to form a tight monolayer, as described above. Five experimental groups were established (n\u0026thinsp;=\u0026thinsp;3 per group): Caco-2 infected with KPN234, Caco-2 infected with KPN234\u003cem\u003eΔiucA\u003c/em\u003e, Uninfected Caco-2 control (PBS-treated), KPN234 pure culture, and KPN234\u003cem\u003eΔiucA\u003c/em\u003e pure culture. For infection groups, approximately 2\u0026times;10⁶ CFU bacteria were added to the apical chamber. Pure culture controls were prepared by inoculating equivalent bacterial numbers into cell culture medium (high-glucose DMEM supplemented with 10% heat-inactivated FBS and 1% non-essential amino acids). All cultures were maintained at 37 ℃ / 5% CO₂ for 4 hours.\u003c/p\u003e\u003cp\u003eFollowing incubation, apical chambers of 24-well transwell plates in three Caco-2 groups were washed with PBS three times, then membranes were excised and flash-frozen in liquid nitrogen immediately. Bacteria in pure culture groups were pelleted by centrifugation (12,000 \u0026times; g, 1 min) and flash-frozen in liquid nitrogen.\u003c/p\u003e\u003cp\u003eTotal RNA of each sample was extracted, and rRNA-depleted libraries were prepared using Ribo-off rRNA Depletion Kit (Vazyme, Cat. NO. N406 for human, Cat. No. N407 for bacteria) and KC-DigitalTM Stranded mRNA Library Prep Kit for Illumina\u0026reg; (Catalog NO. DR08502, Wuhan Seqhealth Co., Ltd. China), which adds a unique molecular identifier (UID) consisting of 12 random bases to the cDNA molecules prior to amplification in order to correct for the preferences and errors generated during the PCR and sequencing process. Libraries (200\u0026ndash;500 bp) were quantified and sequenced on the DNBSEQ-T7 platform (MGI Tech Co., Ltd. China) using PE150 mode.\u003c/p\u003e\u003cp\u003eRaw sequencing data were processed using fastp (v0.23.0) for quality control, followed by UMI-based clustering (95% similarity threshold) to generate consensus sequences. Cleaned reads were aligned to the \u003cem\u003eHomo sapiens\u003c/em\u003e (GRCh38; GCA_000001405.29) and \u003cem\u003eK. pneumoniae\u003c/em\u003e (GCA_017815715.1) genomes using STAR (v2.5.3a) with default parameters. Read counts mapped to exon regions were quantified using featureCounts (Subread-1.5.1, Bioconductor platform) with RPKM normalization. Differential expression analysis was performed using edgeR (v3.28.1) with thresholds of |log2FC|\u0026ge;1 (2-fold change) and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Significant differentially expressed genes were subjected to KEGG pathway enrichment analysis using KOBAS (v2.1.1; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with visualization in R (v4.4.0).\u003c/p\u003e\u003cp\u003eThe raw sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1330555.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e2.1\u003c/strong\u003e Siderophore gene\u003cem\u003e\u0026nbsp;iucA\u003c/em\u003e deletion resulted in a highly significant decrease in hvKP siderophore secretion\u003c/p\u003e\n\u003cp\u003eThe iucA deletion resulted in a significant reduction in siderophore secretion by hvKP, with the \u0026Delta;iucA mutant (KPN234\u0026Delta;iucA) producing only 24.0% of the wild-type (KPN234) levels (\u003cstrong\u003eFig. 1a\u003c/strong\u003e). Notably, the iucA deletion did not significantly affect bacterial growth curve in LB liquid medium (\u003cstrong\u003eFig. 1b\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Deletion of the \u003cem\u003eiucA\u003c/em\u003e gene impairs hvKP infectivity in Caco-2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe invasion/adhesion ratio of wild-type KPN234 was significantly higher than that of the \u003cem\u003e\u0026Delta;iucA\u003c/em\u003e mutant at both 2h and 4h post-infection (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). While KPN234 showed time-dependent enhancement of invasion (2h vs 4h, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), the \u003cem\u003e\u0026Delta;iucA\u003c/em\u003e strain exhibited no significant change (\u003cstrong\u003eFig. 2a\u003c/strong\u003e). Transwell assays revealed that only KPN234 could traverse the Caco-2 monolayer after 4h, with no detectable translocation observed for the \u003cem\u003e\u0026Delta;iucA\u003c/em\u003e mutant (\u003cstrong\u003eFig. 2b\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Wild-type and \u0026Delta;iucA hvKP do not disrupt tight junction integrity in Caco-2 monolayers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTEER measurements demonstrated no significant differences in transepithelial resistance between KPN234-infected, KPN234\u003cem\u003e\u0026Delta;iucA\u003c/em\u003e-infected, and PBS control groups at any time point (\u003cstrong\u003eFig. 3\u003c/strong\u003e). In contrast, EGTA-treated positive controls showed rapid TEER reduction (\u0026gt;80% decrease by 2h post-treatment), approaching baseline levels observed in cell-free inserts. These results indicate that hvKP translocation across Caco-2 monolayers within 4h of infection occurs without compromising tight junction integrity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4\u003c/strong\u003e \u003cstrong\u003e\u003cem\u003eIucA\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;knockout alters the transcriptional profile of hvKP during infection of Caco-2 monolayers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate host-pathogen interactions, we performed dual RNA-seq analysis of hvKP and Caco-2 monolayers at 4 h post-infection. Principal component analysis revealed:\u0026nbsp;after exposure to Caco-2 monolayers, the intra-group variability in all sets increased markedly, reflected by a pronounced separation between individual samples. Moreover, the cell-exposed and medium-only groups were clearly segregated, indicating that hvKP underwent substantial transcriptional reprogramming upon interaction with the Caco-2 monolayer (\u003cstrong\u003eFig. 4a\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eCompared with the control group cultured in medium alone, wild-type hvKP exhibited significant differential expression of 1,446 genes following interaction with the Caco-2 monolayer (\u003cstrong\u003eTable S1\u003c/strong\u003e). Among them, 15 KEGG pathways were enriched for up-regulated genes, whereas 53 pathways were enriched for down-regulated genes (\u003cstrong\u003eTable S2, S3\u003c/strong\u003e). The five pathways with the highest enrichment scores that were exclusively up-regulated were Ribosome, Protein export (SecG, SecM, tatA, tatB), Bacterial secretion system (T2SS: GspK, GspL; T6SS: tssD), Fatty acid biosynthesis, and RNA polymerase (\u003cstrong\u003eFig. 4b-d\u003c/strong\u003e). Conversely, the five most highly enriched pathways showing exclusive down-regulation were C5-Branched dibasic acid metabolism, Lipopolysaccharide biosynthesis, Synthesis and degradation of ketone bodies, Biosynthesis of siderophore group nonribosomal peptides, and Biotin metabolism (\u003cstrong\u003eFig. 4b, c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFollowing infection of Caco-2 monolayers, the \u003cem\u003e\u0026Delta;iucA\u003c/em\u003e mutant exhibited significant downregulation of ribosome biogenesis, ABC transporters, and two-component system pathways compared to wild-type hvKP (\u003cstrong\u003eTable S4;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Fig. 4e\u003c/strong\u003e). These transcriptional alterations correlate with the observed impaired proliferative capacity of the \u003cem\u003e\u0026Delta;iucA\u003c/em\u003e mutant in Caco-2 cells. Concurrently, the mutant upregulated multiple metabolic pathways when exposed to Caco-2 monolayers, including branched-chain amino acid biosynthesis, biotin/CoA production, and glutathione metabolism, demonstrating compensatory metabolic rewiring to enhance nutrient acquisition, redox balance, and environmental adaptation following \u003cem\u003eiucA\u003c/em\u003e loss (\u003cstrong\u003eTable S5; Fig. 4d\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Impact of \u003cem\u003eiucA\u003c/em\u003e deletion on the global transcriptomic response of Caco-2 monolayers to hvKP infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePCA showed that both wild-type and \u003cem\u003e\u0026Delta;iucA\u003c/em\u003e hvKP groups separated clearly from PBS controls, confirming that infection provokes a profound transcriptional reprogramming of the epithelial layer (\u003cstrong\u003eFig. 5a\u003c/strong\u003e). Compared with PBS, Caco-2 monolayers exposed to wild-type hvKP KPN234 displayed 1,851 significant DEGs, with 12 up-regulated KEGG pathways and 21 down-regulated pathways (\u003cstrong\u003eTable S6-8; Fig. 5b\u003c/strong\u003e). The most enriched up-regulated pathway was pentose and glucuronate interconversions, followed by peroxisome, while the TGF-\u0026beta; signaling pathway was also significantly induced (\u003cstrong\u003eFig. 5c\u003c/strong\u003e). Among repressed pathways, protein digestion and absorption showed the strongest enrichment, accompanied by marked down-regulation of oxidative phosphorylation (\u003cstrong\u003eFig. 5d\u003c/strong\u003e). Concurrent upregulation of T2SS (gspK/gspL) and T6SS (tssD) genes in hvKP, coupled with host protein metabolism pathway suppression, implies potential disruption of Caco-2 proteostasis by bacterial effectors (Zhou, \u003cem\u003eet al\u003c/em\u003e., 2022). Finally, the host autophagy pathway was significantly suppressed in infected monolayers (\u003cstrong\u003eFig. 5d, e\u003c/strong\u003e). However, the two bacterial groups clustered together, and DEG analysis revealed only nine genes with |log2FC| \u0026gt; 1 (max = 1.45), indicating that loss of \u003cem\u003eiucA\u003c/em\u003e does not markedly alter host transcriptional signatures (\u003cstrong\u003eTable S9\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussions","content":"\u003cp\u003eSiderophores are critical virulence factors for hvKP, and deletion of siderophore biosynthesis genes leads to a significant reduction in hvKP virulence (Siu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). hvKP produces markedly higher levels of siderophores than cKP, particularly in iron-depleted environments such as human body fluids. However, the role of siderophores in hvKP\u0026rsquo;s breaching of the intestinal mucosal barrier remains incompletely understood.\u003c/p\u003e\u003cp\u003eOur results demonstrate that \u003cem\u003eiucA\u003c/em\u003e knockout reduces total siderophore secretion by nearly 80% in basal bacterial culture media. Russo et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) reported even more pronounced reductions\u0026mdash;95% in ascites, 94% in urine, and 100% in serum\u0026mdash;when \u003cem\u003eiucA\u003c/em\u003e was mutated, suggesting that nutrient-rich media may partially mask the impact of siderophore deficiency. Hsu \u003cem\u003eet al.\u003c/em\u003e (2014) showed that systemic infection-causing KP strains of the K2 capsular serotype can adhere to, invade, and translocate across Caco-2 cell monolayers. Our study reveals that the K20 serotype hvKP strain KPN234 (wild-type) shares these capabilities, though with comparatively weaker adhesion/invasion efficiency (\u0026lt;\u0026thinsp;0.6% vs. 1.46% \u0026plusmn; 0.18% reported by Hsu \u003cem\u003eet al.\u003c/em\u003e). This discrepancy may stem from KPN234\u0026rsquo;s hypermucoviscous phenotype, as studies suggest mucoidity negatively correlates with bacterial invasiveness (Xu, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Transepithelial electrical resistance (TEER) assays confirmed that hvKP traverses Caco-2 monolayers without disrupting tight junctions, consistent with findings of Hsu \u003cem\u003eet al.\u003c/em\u003e(2014). However, at the transcriptional level, we still observed significant downregulation of tight junction pathways in Caco-2 cell monolayers following hvKP infection (Table \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e), suggesting that prolonged interaction with hvKP may ultimately compromise epithelial barrier integrity.\u003c/p\u003e\u003cp\u003eAlthough \u003cem\u003eiucA\u003c/em\u003e deletion did not impair hvKP\u0026rsquo;s in vitro proliferation, it significantly attenuated adhesion, invasion, and monolayer translocation. Parallel observations were reported by Deng \u003cem\u003eet al.\u003c/em\u003e(2022): \u003cem\u003eiucA\u003c/em\u003e deficiency in uropathogenic \u003cem\u003eE. coli\u003c/em\u003e (UPEC) reduced adhesion to bladder epithelial cells, impaired bladder colonization in mice, and hindered urothelial transmigration. Notably, \u003cem\u003eiucA\u003c/em\u003e knockout also downregulated quorum sensing (QS) pathway (including LuxS, QseC, and 12 other genes) in infected Caco-2 monolayers\u0026mdash;a system tightly linked to biofilm formation (Table \u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003e). Prior work has reported that siderophore production positively regulates biofilm development (Sadiq \u003cem\u003eet al\u003c/em\u003e., 2024).\u003c/p\u003e\u003cp\u003eWhile \u003cem\u003eiucA\u003c/em\u003e knockout caused an 80% decline in siderophore secretion (MM9 medium), it did not alter the transcriptional profile of Caco-2 monolayers. Thus, \u003cem\u003eiucA\u003c/em\u003e\u0026rsquo;s influence on infectivity likely stems from its regulation of bacterial physiology, including metabolic and signaling adaptations. This contrasts with in vivo findings: Wu \u003cem\u003eet al\u003c/em\u003e. demonstrated that \u003cem\u003eiucA\u003c/em\u003e-deficient hvKP induced less hepatic oxidative stress and tissue damage in mice than the wild-type (Wu, et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Siderophores typically affect host cells indirectly\u0026mdash;e.g., by sequestering iron to disrupt host oxidative burst capacity (Marchetti, et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) or modulating immune responses via other virulence factors (Sheldon, et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The divergence between our in vitro and published in vivo results may reflect fundamental differences in iron dynamics: systemic immune responses (e.g., neutrophil recruitment or macrophage polarization) likely amplify siderophore-mediated pathology in vivo, whereas monoculture models lack such multicellular crosstalk. Future in vivo intestinal infection models should elucidate \u003cem\u003eiucA\u003c/em\u003e's mechanistic role in hvKP gut translocation.\u003c/p\u003e\u003cp\u003eTranscriptomic analysis revealed that hvKP infection significantly activates the pentose and glucuronate interconversions pathway in Caco-2 cell monolayers. This metabolic pathway converts pentoses into glucuronate, which subsequently enters the pentose phosphate pathway (PPP) to facilitate NADPH generation (Vo, et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), suggesting that hvKP may reprogram the host metabolic network to meet its energy and metabolic precursor demands. Notably, multiple pathogens including \u003cem\u003eCryptococcus neoformans\u003c/em\u003e, \u003cem\u003eChlamydia\u003c/em\u003e, and \u003cem\u003ePlasmodium\u003c/em\u003e species have been shown to hijack the host PPP to acquire essential metabolites (Oyelade, et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Price, et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Yang, et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).. The specific upregulation of this pathway in Caco-2 cells observed in our study likely represents a host metabolic adaptive response to hvKP infection, while also implying that hvKP may similarly exploit host metabolic resources to sustain its infectious progression.\u003c/p\u003e\u003cp\u003eWe also detected significant upregulation of the TGF-β signaling pathway in infected cell monolayers. As a well-characterized immunosuppressive pathway, TGF-β activation typically facilitates pathogen survival during infection (Deng et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which is consistent with previous reports of its induction during classical KP (cKP) infection (Liu \u003cem\u003eet al\u003c/em\u003e., 2022). Surprisingly, our data revealed marked downregulation of autophagy pathways in hvKP-infected Caco-2 monolayers. This finding appears paradoxical because: (1) impaired autophagy has been shown to increase intestinal barrier permeability (Wu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e); and (2) most enteric pathogens (e.g., Salmonella, Enterococcus, Shigella) actually activate epithelial autophagy as part of host defense mechanisms \u003cem\u003eet al\u003c/em\u003e., 2019). While KP infection has been reported to induce autophagy in macrophages and alveolar epithelial cells (Shi et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), our results indicate that hvKP may employ a distinct strategy by suppressing autophagy specifically in intestinal epithelial cells. Considering the reported tissue-specific adaptation of KP virulence mechanisms in a previous study (Holmes et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the observed autophagy inhibition in gut epithelium may suggest a unique host\u0026ndash;pathogen interaction that merits further investigation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by Hundred Young Researchers Grant Program [grant number GDMUD2023004] and Dongguan Science and Technology of Social Development Program [grant number 20231800936202].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTingting Zhi\u003c/strong\u003e designed the experiments, wrote the original draft, and acquired funding\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Chunjie Zhu\u0026nbsp;\u003c/strong\u003edesigned the experiments,\u0026nbsp;analyzed the data, and acquired funding\u003cstrong\u003e. Zi Caiqin\u0026nbsp;\u003c/strong\u003eperformed the experiments\u0026nbsp;and analyzed the data.\u003cstrong\u003e\u0026nbsp;Jinxiu Xie\u0026nbsp;\u003c/strong\u003eanalyzed the data\u0026nbsp;and visualization of the data\u003cstrong\u003e. Ruijun Lao\u0026nbsp;\u003c/strong\u003eperformed the experiments.\u003cstrong\u003e\u0026nbsp;Liping Wang\u0026nbsp;\u003c/strong\u003eprovided expert technical guidance on cell culture methodologies\u003cstrong\u003e. Jiangmei Gao\u0026nbsp;\u003c/strong\u003eprovided expert advice on data interpretation\u003cstrong\u003e. Na Mi\u0026nbsp;\u003c/strong\u003econceived the study andsupervised the research. \u003cstrong\u003eZuguo Zhao\u0026nbsp;\u003c/strong\u003esupervised the research, reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;interaction transcriptomics\u0026nbsp;datasets of this article are available in the NCBI SRA under BioProject accession number PRJNA1330555 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1330555).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBailey, D. C., Alexander, E., Rice, M. R., Drake, E. J., Mydy, L. S., Aldrich, C. C., \u0026amp; Gulick, A. M. (2018). Structural and functional delineation of aerobactin biosynthesis in hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. \u003cem\u003eJ Biol Chem\u003c/em\u003e\u003cem\u003e, 293\u003c/em\u003e(20), 7841-52. doi: 10.1074/jbc.RA118.002798\u003c/li\u003e\n \u003cli\u003eBaker, N. T., \u0026amp; Graham, L. L. (2010). Campylobacter fetus translocation across Caco-2 cell monolayers. \u003cem\u003eMicrob Pathog, 49\u003c/em\u003e(5), 260-72. doi: 10.1016/j.micpath.2010.06.008\u003c/li\u003e\n \u003cli\u003eChoby, J. E., Howard-Anderson, J., \u0026amp; Weiss, D. S. (2020). Hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e - clinical and molecular perspectives. \u003cem\u003eJ Intern Med, 287\u003c/em\u003e(3), 283-300. doi: 10.1111/joim.13007\u003c/li\u003e\n \u003cli\u003eDeng, C., Kang, J. L., Lin, M. S., \u0026amp; Lin, Y. F.(2022). The effect of iucA gene deletion on the proliferation, adhesion, invasion and colonization ability of Uropathogenic \u003cem\u003eE. coli\u003c/em\u003e. \u003cem\u003eActa Univ Med Anhui\u003c/em\u003e, \u003cem\u003e57\u003c/em\u003e(02), 177-81. doi: 10.19405/j.cnki.issn1000-1492.2022.02.003.\u003c/li\u003e\n \u003cli\u003eDeng, Z., Fan, T., Xiao, C., Tian, H., Zheng, Y., Li, C., \u0026amp; He, J. (2024). TGF-\u0026beta; signaling in health, disease and therapeutics. \u003cem\u003eSignal Transduct Target Ther\u003c/em\u003e\u003cem\u003e, 9\u003c/em\u003e(1), 61. doi: 10.1038/s41392-024-01764-w\u003c/li\u003e\n \u003cli\u003eFinlay, B. B., \u0026amp; Falkow, S. (1990). Salmonella interactions with polarized human intestinal Caco-2 epithelial cells. \u003cem\u003eJ Infect Dis, 162\u003c/em\u003e(5), 1096-106. doi: 10.1093/infdis/162.5.1096\u003c/li\u003e\n \u003cli\u003eFung, C. P., Lin, Y. T., Lin, J. C., Chen, T. L., Yeh, K. M., Chang, F. Y., Chuang, H. C., Wu, H. S., Tseng, C. P., \u0026amp; Siu, L. K. (2012). \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e in gastrointestinal tract and pyogenic liver abscess. \u003cem\u003eEmerg Infect Dis, 18\u003c/em\u003e(8), 1322-5. doi: 10.3201/eid1808.111053\u003c/li\u003e\n \u003cli\u003eHaq, S., Grondin, J., Banskota, S., \u0026amp; Khan, W. I. (2019). Autophagy: roles in intestinal mucosal homeostasis and inflammation. \u003cem\u003eJ Biomed Sci, 26\u003c/em\u003e(1), 19. doi: 10.1186/s12929-019-0512-2\u003c/li\u003e\n \u003cli\u003eHolmes,\u0026nbsp;C. L., Wilcox, A. E., Forsyth, V., Smith, S. N., Moricz, B. S., Unverdorben, L. V., Mason, S., Wu, W., Zhao, L., Mobley, H. L. T., \u0026amp; Bachman, M. A. (2023). \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e causes bacteremia using factors that mediate tissue-specific fitness and resistance to oxidative stress. \u003cem\u003ePLoS Pathog, 19\u003c/em\u003e(7), e1011233. doi: 10.1371/journal.ppat.1011233\u003c/li\u003e\n \u003cli\u003eHsu, C. R., Pan, Y. J., Liu, J. Y., Chen, C. T., Lin, T. L., \u0026amp; Wang, J. T. (2015). \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e translocates across the intestinal epithelium via Rho GTPase- and phosphatidylinositol 3-kinase/Akt-dependent cell invasion. \u003cem\u003eInfect Immun, 83\u003c/em\u003e(2), 769-79. doi: 10.1128/iai.02345-14\u003c/li\u003e\n \u003cli\u003eKocsis, B. (2023). Hypervirulent\u003cem\u003e\u0026nbsp;Klebsiella pneumoniae\u003c/em\u003e: An update on epidemiology, detection and antibiotic resistance. \u003cem\u003eActa Microbiol Immunol Hung\u003c/em\u003e\u003cem\u003e, 70\u003c/em\u003e(4), 278-87. doi: 10.1556/030.2023.02186\u003c/li\u003e\n \u003cli\u003eLan, P., Shi, Q., Zhang, P., Chen, Y., Yan, R., Hua, X., Jiang, Y., Zhou, J., \u0026amp; Yu, Y. (2020). Core Genome Allelic Profiles of Clinical \u003cem\u003eKlebsiella pneumoniae\u0026nbsp;\u003c/em\u003eStrains Using a Random Forest Algorithm Based on Multilocus Sequence Typing Scheme for Hypervirulence Analysis. \u003cem\u003eJ Infect Dis, 221\u003c/em\u003e(Suppl 2), S263-71. doi: 10.1093/infdis/jiz562\u003c/li\u003e\n \u003cli\u003eLee, C. R., Lee, J. H., Park, K. S., Jeon, J. H., Kim, Y. B., Cha, C. J., Jeong, B. C., \u0026amp; Lee, S. H. (2017). Antimicrobial Resistance of Hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e: Epidemiology, Hypervirulence-Associated Determinants, and Resistance Mechanisms. \u003cem\u003eFront Cell Infect Microbiol., 7,\u0026nbsp;\u003c/em\u003e483. doi: 10.3389/fcimb.2017.00483\u003c/li\u003e\n \u003cli\u003eLee, I. R., Molton, J. S., Wyres, K. L., Gorrie, C., Wong, J., Hoh, C. H., Teo, J., Kalimuddin, S., Lye, D. C., Archuleta, S., Holt, K. E., \u0026amp; Gan, Y. H. (2016). Differential host susceptibility and bacterial virulence factors driving Klebsiella liver abscess in an ethnically diverse population. \u003cem\u003eSci Rep, 6\u003c/em\u003e, 29316. doi: 10.1038/srep29316\u003c/li\u003e\n \u003cli\u003eLiu, C., \u0026amp; Guo, J. (2019). Hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (hypermucoviscous and aerobactin positive) infection over 6 years in the elderly in China: antimicrobial resistance patterns, molecular epidemiology and risk factor. \u003cem\u003eAnn Clin Microbiol Antimicrob, 18\u003c/em\u003e(1), 4. doi: 10.1186/s12941-018-0302-9\u003c/li\u003e\n \u003cli\u003eMarchetti, M., De Bei, O., Bettati, S., Campanini, B., Kovachka, S., Gianquinto, E., Spyrakis, F., \u0026amp; Ronda, L. (2020).\u0026nbsp;Iron Metabolism at the Interface between Host and Pathogen: From Nutritional Immunity to Antibacterial Development. \u003cem\u003eInt J Mol Sci, 21\u003c/em\u003e(6), 2415. doi: 10.3390/ijms21062145\u003c/li\u003e\n \u003cli\u003eNguyen, Q., Nguyen, Y. T. P., Ha, T. T., Tran, D. T. N., Voong, P. V., Chau, V., Nguyen, P. L. N., Le, N. T. Q., Nguyen, L. P. H., Nguyen, T. T. N., Trinh, T. V., Carrique-Mas J. J., Baker, S., Thwaites, G., Rabaa, M. A., Choisy, M., Chung, H. T., \u0026amp; Pham, D. T. (2024). Genomic insights unveil the plasmid transfer mechanism and epidemiology of hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e in Vietnam. \u003cem\u003eNat Commun, 15\u003c/em\u003e(1), 4187. doi: 10.1038/s41467-024-48206-3\u003c/li\u003e\n \u003cli\u003eOyelade, J., Isewon, I., Aromolaran, O., Uwoghiren, E., Dokunmu, T., Rotimi, S., Aworunse, O., Obembe, O., \u0026amp; Adebiyi, E. (2019). Computational Identification of Metabolic Pathways of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e using thek-Shortest Path Algorithm. \u003cem\u003eInt J Genomics, 2019\u003c/em\u003e, 1-13. doi: 10.1155/2019/1750291\u003c/li\u003e\n \u003cli\u003ePan, Y. J., Fang, H. C., Yang, H. C., Lin, T. L., Hsieh, P. F., Tsai, F. C., Keynan, Y., \u0026amp; Wang, J. T. (2008). Capsular polysaccharide synthesis regions in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e serotype K57 and a new capsular serotype. \u003cem\u003eJ Clin Microbiol, 46\u003c/em\u003e(7), 2231-40. doi: 10.1128/jcm.01716-07\u003c/li\u003e\n \u003cli\u003ePrice, M. S., Betancourt-Quiroz, M., Price, J. L., Toffaletti, D. L., Vora, H., Hu, G., Kronstad, J. W., Perfect, J. R., \u0026amp; Dromer, F. (2011). Cryptococcus neoformans Requires a Functional Glycolytic Pathway for Disease but Not Persistence in the Host. \u003cem\u003emBio, 2\u003c/em\u003e(3), e00103-11. doi: 10.1128/mBio.00103-11\u003c/li\u003e\n \u003cli\u003eRusso, T. A., Olson, R., MacDonald, U., Beanan, J., \u0026amp; Davidson, B. A. (2015). Aerobactin, but not yersiniabactin, salmochelin, or enterobactin, enables the growth/survival of hypervirulent (hypermucoviscous) \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e ex vivo and in vivo. \u003cem\u003eInfect Immun, 83\u003c/em\u003e(8), 3325-33. doi: 10.1128/iai.00430-15\u003c/li\u003e\n \u003cli\u003eRusso, T. A., Olson, R., Macdonald, U., Metzger, D., Maltese, L. M., Drake, E. J., \u0026amp; Gulick, A. M. (2014). Aerobactin mediates virulence and accounts for increased siderophore production under iron-limiting conditions by hypervirulent (hypermucoviscous) \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. \u003cem\u003eInfect Immun, 82\u003c/em\u003e(6), 2356-67. doi: 10.1128/iai.01667-13\u003c/li\u003e\n \u003cli\u003eSadiq\u0026nbsp;Aniz, E. H., \u0026amp; Jassim, Y. A. (2024). Anticancer activity of pyoverdine (PVD) producing by antibiotic-resistant\u003cem\u003e\u0026nbsp;Pseudomonas aeruginosa\u003c/em\u003e isolated from burn and wound infections.\u003cem\u003e\u0026nbsp;J Appl Nat Sci, 16\u003c/em\u003e(2). doi: 10.31018/jans.v16i2.5506\u003c/li\u003e\n \u003cli\u003eSheldon, J. R., Heinrichs, D. E., \u0026amp; Whitfield, C. (2015). Recent developments in understanding the iron acquisition strategies of gram positive pathogens. \u003cem\u003eFEMS Microbiol Rev, 39\u003c/em\u003e(4), 592-630. doi: 10.1093/femsre/fuv009\u003c/li\u003e\n \u003cli\u003eShi, Z. Y., Yin, X., Hou, X., Wang, L. Q., Li, G., Wang, J., Wang, W., Zhang, Y., Li, S., \u0026amp; Jia, W. (2023). Mechanism of Notch Signal Regulating Alveolar Epithelial Cell Autophagy after Infection with \u003cem\u003eKlebsiella Pneumoniae\u003c/em\u003e. \u003cem\u003eClin Lab, 69\u003c/em\u003e(5). doi:\u0026nbsp;10.7754/Clin.Lab.2022.211203\u003c/li\u003e\n \u003cli\u003eSiu, L. K., Fung, C. P., Chang, F. Y., Lee, N., Yeh, K. M., Koh, T. H., \u0026amp; Ip, M. (2011). Molecular typing and virulence analysis of serotype K1 \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e strains isolated from liver abscess patients and stool samples from noninfectious subjects in Hong Kong, Singapore, and Taiwan. \u003cem\u003eJ Clin Microbiol, 49\u003c/em\u003e(11), 3761-5. doi: 10.1128/jcm.00977-11\u003c/li\u003e\n \u003cli\u003eSpellberg, B., Russo, T. A., Shon, A. S., Beanan, J. M., Olson, R., MacDonald, U., Pomakov, A. O., \u0026amp; Visitacion, M. P. (2011). Hypervirulent \u003cem\u003eK. Pneumoniae\u003c/em\u003e Secretes More and More Active Iron-Acquisition Molecules than \u0026ldquo;Classical\u0026rdquo; \u003cem\u003eK. Pneumoniae\u003c/em\u003e Thereby Enhancing its Virulence. \u003cem\u003ePLoS ONE, 6\u003c/em\u003e(10), e26734. doi: 10.1371/journal.pone.0026734\u003c/li\u003e\n \u003cli\u003eVo, P. N. L., Lee, H. M., Ren, J., \u0026amp; Na, D. (2021). Optimized expression of Hfq protein increases \u003cem\u003eEscherichia coli\u003c/em\u003e growth. \u003cem\u003eJ Biol Eng, 15\u003c/em\u003e(1). doi: 10.1186/s13036-021-00260-x\u003c/li\u003e\n \u003cli\u003eWang, X., Bi, C., Xin, X., Zhang, M., Fu, H., Lan, L., Wang, M., \u0026amp; Yan, Z. (2023). Pyroptosis, apoptosis, and autophagy are involved in infection induced by two clinical \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e isolates with different virulence. \u003cem\u003eFront Cell Infect Microbiol, 13\u003c/em\u003e, 1165609. doi: 10.3389/fcimb.2023.1165609\u003c/li\u003e\n \u003cli\u003eWu, J., Chen, J., Wang, Y., Meng, Q., \u0026amp; Zhao, J. (2022). Siderophore iucA of hypermucoviscous \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e promotes liver damage in mice by inducing oxidative stress. \u003cem\u003eBiochem Biophys Rep, 32\u003c/em\u003e, 101376. doi: 10.1016/j.bbrep.2022.101376\u003c/li\u003e\n \u003cli\u003eWu, S., Zhang, Y. G., Lu, R., Xia, Y., Zhou, D., Petrof, E. O., Claud, E. C., Chen, D., Chang, E. B., Carmeliet, G., \u0026amp; Sun, J. (2015). Intestinal epithelial vitamin D receptor deletion leads to defective autophagy in colitis. \u003cem\u003eGut, 64\u003c/em\u003e(7), 1082-94. doi: 10.1136/gutjnl-2014-307436\u003c/li\u003e\n \u003cli\u003eXu, Q., Yang, X., Chan, E. W. C., \u0026amp; Chen, S. (2021). The hypermucoviscosity of hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e confers the ability to evade neutrophil-mediated phagocytosis. \u003cem\u003eVirulence, 12\u003c/em\u003e(1), 2050-9. doi: 10.1080/21505594.2021.1960101\u003c/li\u003e\n \u003cli\u003eYang, L., Liu, C., Zhao, W., He, C., Ding, J., Dai, R., Xu, K., Xiao, L., Luo, L., Liu, S., Li, W., \u0026amp; Meng, H. (2018). Impaired Autophagy in Intestinal Epithelial Cells Alters Gut Microbiota and Host Immune Responses.\u0026nbsp;\u003cem\u003eAppl Environ Microbiol, 84\u003c/em\u003e(18), e00880-18. doi: 10.1128/aem.00880-18\u003c/li\u003e\n \u003cli\u003eYang, S., Zeng, J., Yu, J., Sun, R., Tuo, Y., \u0026amp; Bai, H. (2024). Insights into Chlamydia Development and Host Cells Response.\u0026nbsp;\u003cem\u003eMicroorganisms,\u003c/em\u003e\u003cem\u003e\u0026nbsp;12\u003c/em\u003e(7), 1302. doi: 10.3390/microorganisms12071302\u003c/li\u003e\n \u003cli\u003eZhou, H., Beltr\u0026aacute;n, J. F., \u0026amp; Brito, I. L. (2022). Host-microbiome protein-protein interactions capture disease-relevant pathways. \u003cem\u003eGenome Biol, 23\u003c/em\u003e(1), 72. doi: 10.1186/s13059-022-02643-9\u003c/li\u003e\n \u003cli\u003eZi, C., Yang, S., Fu, X. F., Wang, W. Q., Luo, Y., Zhang, J., Guo, W. T., Wang, H. P., Li, L., Liang, X. Y., Mi, N., Zhi, T. T, \u0026amp; Zhao, Z. G. (2023). An efficient method for knocking out genes on the virulence plasmid of hypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. \u003cem\u003eNew Microbiol, 46\u003c/em\u003e(2), 186-95. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hypervirulent Klebsiella pneumoniae, Caco-2, infection, siderophore, interaction transcriptomics","lastPublishedDoi":"10.21203/rs.3.rs-7689359/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7689359/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHypervirulent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (hvKP) causes lethal systemic infections following intestinal colonization, but the mechanisms underlying its translocation across the intestinal barrier remain unclear. In this study, we used Caco-2 cells as a model to investigate the role of the aerobactin siderophore-encoding gene \u003cem\u003eiucA\u003c/em\u003ein the invasion process of hvKP. We observed that the wild-type (WT) hvKP strain exhibited significantly higher invasion rates than the \u003cem\u003eiucA\u003c/em\u003e-deletion mutant (\u003cem\u003eΔiucA\u003c/em\u003e) at 2 and 4 hours post-infection (P \u0026lt; 0.05). Furthermore, only the WT strain penetrated dense Caco-2 monolayers, indicating that \u003cem\u003eiucA\u003c/em\u003e is essential for traversing the intestinal mucosal barrier. Interaction transcriptomics revealed distinct host-pathogen dynamics: hvKP infection significantly activated pathways related to protein export and the bacterial secretion system; Caco-2 cells upregulated pentose/glucuronate interconversions, peroxisome activity, and TGF-β signaling but downregulated protein metabolism and autophagy; The transcriptional profile of Caco-2 monolayers was minimally altered by the presence or absence of \u003cem\u003eiucA\u003c/em\u003e, suggesting that aerobactin’s role is primarily bacterial-centric. This study reveals the central role of \u003cem\u003eiucA\u003c/em\u003e-mediated aerobactin synthesis in the invasion of the intestinal mucosal barrier by hvKP, providing important insights into the interaction between hvKP and intestinal epithelial cells.\u003c/p\u003e","manuscriptTitle":"IucA gene contributes to the infectivity of hypervirulent Klebsiella pneumoniae in Caco-2 cell models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-09 05:03:51","doi":"10.21203/rs.3.rs-7689359/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c1bba419-1477-4acc-aace-14c2b95e3c60","owner":[],"postedDate":"October 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-22T04:38:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-09 05:03:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7689359","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7689359","identity":"rs-7689359","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}