Functional insights into nucleoside diphosphate kinases encoded by two ndk paralogs in Waddlia chondrophila

Functional insights into nucleoside diphosphate kinases encoded by two ndk paralogs in Waddlia chondrophila | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Functional insights into nucleoside diphosphate kinases encoded by two ndk paralogs in Waddlia chondrophila Giti Ghazi-Soltani, Carole Kebbi-Beghdadi, Simone E. Adams, Gilbert Greub This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8206315/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 The Chlamydiota phylum consists of obligate intracellular bacteria, including well-known pathogens and emerging environmental species, with diverse host ranges and metabolic capabilities. Among these bacteria, the gene, which encodes nucleoside diphosphate kinase ( ndk ), is present in variable copy numbers. While most chlamydial species carry a single copy of ndk , some species have two copies. In W. chondrophila , the two Ndk proteins encoded by ndk paralogs retain conserved kinase motifs but differ in subcellular localization, suggesting divergent functional roles. According to localization studies performed inheterologous expression systems, WcNdk1 is confined to the inclusion and probably supports nucleotide metabolism, while WcNdk2 localizes to the host nucleus, perinuclear space, and Golgi apparatus, suggesting involvement in host interaction. Azidothymidine (AZT), a known Ndk inhibitor, impaired W. chondrophila growth , potentially through inhibition of WcNdk2 . However, the lack of genetic tools and the absence of in vitro enzymatic assays currently limit definitive functional conclusions. Our data suggest potential functions for Ndks in W. chondrophila, providing a foundation for future studies on Ndk-mediated interactions between this pathogen and its host. Biological sciences/Biochemistry Biological sciences/Evolution Biological sciences/Microbiology Biological sciences/Molecular biology Biological sciences/Structural biology Waddlia chondrophila Nucleoside diphosphate kinase (Ndk) AZT inhibition Subcellular localization Protein trafficking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION The Chlamydiota phylum encompasses several families of obligate intracellular bacteria, including six main families: Chlamydiaceae , Parachlamydiaceae , Waddliaceae , Simkaniaceae , Rhabdochlamydiaceae , and Criblamydiaceae . Members of these families share a biphasic developmental cycle, which reflects their adaptation to intracellular environments and reliance on host cell machinery for survival. W. chondrophila belongs to the family Waddliaceae and is known as an abortigenic agent in ruminants. It was first isolated from an aborted bovine fetus 1,2 and serological studies showed a strong association between anti- Waddlia antibodies and bovine abortion 3 . In humans, W. chondrophila seropositivity is significantly associated with adverse pregnancy outcomes 4–6 . This pathogen was also detected in respiratory tract samples of patients with pneumonia and children with bronchiolitis 7,8 . The W. chondrophila developmental cycle is biphasic and is divided into three major phases 9,10 . In the early phase (0–8 hours post-infection, hpi) elementary bodies (EBs) enter in host cells and differentiate into reticulate bodies (RBs). This phase is followed by a proliferation phase (8–24 hpi) during which the number of RBs increases exponentially through binary fission. In the late phase (24–48 hpi), RBs revert into EBs and lysis of the host cell releases infectious progeny. Under environmental or antibiotic stress, the developmental cycle is arrested leading to an enlarged, non-dividing RB-like form called Aberrant Body (AB) 11 . While W. chondrophila significantly impacts both animal and human health, the specific molecular and genomic pathways, particularly those involving developmental and regulatory proteins, remain largely unexplored. Functional characterization of proteins, such as nucleoside diphosphate kinase (Ndk), could help us to better understand the biology and pathogenicity of this pathogen and its adaptation inside host cells. Ndk is a highly conserved enzyme that plays a central role in maintaining nucleotide pools within both bacterial and eukaryotic cells. The maintenance of intracellular NTP pools is vital for the survival and proliferation of all living organisms. Ndk regulates nucleotide pools through its autophosphorylation and phosphotransferase activities. This ensures that NTP and dNTP levels remain balanced for optimal cellular function. Beyond its housekeeping functions, Ndk is implicated in various cellular processes, including cell differentiation and development in eukaryotes and prokaryotes 12–15 . Additionally, Ndk is involved in the regulation of gene expression 16–18 . Due to its ability to bind ATP, Ndk is involved in inhibiting extracellular ATP (eATP) and disrupting purinergic signaling in immune and inflammatory responses 19–21 . We previously reported transcriptomic data for W. chondrophila comparing genes expression in RBs at 24 hpi and EBs at 72 hpi 22 . In that dataset, both ndk genes were differentially expressed between the two developmental stages, each showing a fold change of 0.3 in EBs compared with RBs. Despite the well-established functions of Ndk across various organisms, its specific function in the Chlamydiota remains uncharacterized. Based on these observations, we hypothesized that this enzyme could play an important role in the development and pathogenesis of W. chondrophila . In this study, we characterize the two Ndk proteins in this organism to better understand their potential role in the bacterium’s development. This study provides an overall view of Ndk proteins in the Chlamydiota phylum and offers functional insights into the Ndks proteins of W. chondrophila . Our results pave the way for more detailed research once genetic tools become available for W. chondrophila . RESULTS Evolution of the ndk operon in the Chlamydiota phylum Similar to other organisms, all bacteria of the Chlamydiota phylum possess at least one copy of the ndk gene. A second copy ( ndk2 ) is present in members of the Parachlamydiaceae , Waddliaceae , and Criblamydiaceae families (Fig. 1 A). Interestingly, ndk2 forms an operon with the ancestral ndk1 gene. In most species, this operon is further associated with pabA , which encodes para-aminobenzoate synthase and is involved in the folate biosynthesis pathway. Comparison of gene-based and species-based phylogenetic trees (Fig. 1 B, C) suggests that the duplication of ndk2 most likely originated from a single gene duplication event in the common ancestor of the Parachlamydiaceae , Waddliaceae , and Criblamydiaceae . However, a scenario of ancestral duplication with subsequent loss in Simkaniaceae and Chlamydiaceae cannot be excluded, although it is less parsimonious. High sequence and structural conservation of Ndk across species The multiple sequence alignment shows a high degree of sequence conservation of Ndk proteins from various bacterial and eukaryotic species (Fig. 2 A). Specifically, the His-Gly-Ser-Asp (HGSD) motif, which is the enzyme’s active site 23 , is well conserved across all aligned sequences. The highly conserved histidine residue inside this motif transiently receives the phosphate group during autophosphorylation reaction and is, therefore, critical for the catalytic function of Ndk. In addition to the HGSD motif, other conserved residues are observed in regions associated with nucleotide binding and structural stability, many of which are enriched in hydrophobic amino acids. These residues likely contribute to the proper folding and enzymatic efficiency of Ndk. W. chondrophila contains two copies of ndk : wcndk1 ( locus wcw_1543 ) and wcndk2 (locus wcw_1545 ), with a sequence similarity of 54% (Fig. 2 B). The most striking difference between the two W. chondrophila ndk paralogs is the presence of a predicted signal peptide of 17 amino acids in WcNdk2, which is absent in WcNdk1. The sequence similarity matrix of W. chondrophila Ndk proteins, WcNdk1 and WcNdk2, and their counterparts from other bacterial and eukaryotic species shows differing degrees of conservation as shown in Fig. 2 B. WcNdk1 shares the highest sequence identity with E. coli Ndk (EcNdk) and Chlamydia trachomatis Ndk (CtNdk) (67% and 66%, respectively). In contrast, WcNdk2 exhibits slightly lower sequence similarity across species, which may indicate functional divergence of the two Ndks following gene duplication. Interestingly, both W. chondrophila Ndk homologs display relatively high sequence identity (~ 50%) with human Ndk (HsNdk), which suggests the conservation of core functional domains across vast evolutionary distance. Sequence similarity matrix of Ndk proteins across different species is shown in supplementary Table S2 . WcNdk1 has 143 residues forming a polypeptide chain and adopting a very similar fold to Ndk proteins from other origins, including human Ndk 24,25 (Fig. 2 C). One Ndk unit has α/β domains comprising a four-stranded antiparallel β-sheet and two connecting α-helices. This high degree of sequence and structural conservation between eukaryotes and pathogens may enable them to interfere with host cellular processes by mimicking host Ndk. Expression profile of W. chondrophila Ndks during infection Temporal expression levels of genes and proteins across different developmental stages may help us understand their stage-specific roles and regulatory mechanisms. Since each of these stages has unique biological features, we can indirectly infer a protein’s function by mapping both transcript and protein levels across these developmental stages. We quantified the expression profiles of the W. chondrophila ndks , at both the transcript and protein level during McCoy cell infection (Fig. 3 ). wcndk1 and wcndk2 transcripts were highly abundant at early time points (3–8 hpi) as revealed by RT-qPCR, then the mRNA level declined sharply to a minimum at 32 hpi, with a moderate surge toward the end of the cycle (48 hpi) (Fig. 3 A). In parallel, protein expression profiles of WcNdk1and WcNdk2 were analyzed using antibodies specifically raised against each protein. No WcNdk1 or WcNdk2 protein could be detected by Western blot of the W. chondrophila -infected cells at 3 to 8 hpi (Fig. 3 B, C). Both proteins became detectable by 24 hpi by Western blot and reached their highest signal intensity at this time point after normalization to bacterial number, followed by a drop to roughly 30% of that level by 48 hpi. To further assess protein expression at early stages, we performed immunofluorescence microscopy. In contrast to Western blot results, WcNdk1 and WcNdk2 signals were already detectable from 0 to 16 hpi, colocalizing with W. chondrophila (Supplementary Figure S1 ). Together, these results suggests that both WcNdk1 and WcNdk2 are expressed throughout the developmental cycle, with an early transcriptional peak. While protein expression begins early, overall accumulation or detection sensitivity by Western blot peaks around 24 hpi, followed by a decline toward the end of the cycle. Subcellular localization of WcNdk1 and WcNdk2 in C. trachomatis heterologous expression While signal peptides are typically associated with protein secretion, previous studies have demonstrated that Ndk can be secreted in certain bacterial species despite lacking a canonical signal sequence 26–28 . Moreover, the secretion of both WcNdk1 and WcNdk2 into the host cell cytoplasm was experimentally shown 29 , suggesting that WcNdk2 signal peptide may not contribute to secretion. The presence of a signal peptide on WcNdk2 suggests that this protein may be translocated to a specific subcellular compartment, giving it a biological role distinct from WcNdk1. To assess this possibility, we overexpressed a V5-tagged versions of WcNdk1 and WcNdk2 in C. trachomatis using the inducible shuttle vector pGL2 and studied their subcellular localization by immunofluorescence and confocal microscopy (Fig. 4 ). In this heterologous expression assay, WcNdk1 was exclusively detected inside the inclusion, closely associated with C. trachomatis , and no signal was observed in the host cell cytoplasm. Although this does not rule out secretion of WcNdk1, it suggests limited accumulation or detection outside the inclusion. In contrast, WcNdk2 was detected in both chlamydial inclusions and the host-cell nucleus, implying that it may be transported into the nucleus. To further investigate the nuclear localization of WcNdk2, we cloned the gene into the pBOMBL vector 30 and overexpressed the protein in C. trachomatis . Upon infection of McCoy cells with the transformed bacteria, inclusions appeared smaller compared to controls. Due to these growth defects, reliable localization of WcNdk2 could not be realized in this system. This vector-associated toxicity likely reflects bacterial stress caused by excessive or mis-regulated expression of WcNdk2, similar to the overexpression-associated toxicity reported for type III effectors in Pseudomonas aeruginosa 31 . Consistently, WcNdk2 expression was also not detected in the Yersinia enterocolitica type III secretion assay, suggesting poor tolerance or instability of the protein in this system. Subcellular localization of WcNdk1 and WcNdk2 in transfected HEK293T cells To further confirm the observed subcellular localization of WcNdk1 and WcNdk2 in another heterologous system, we transiently expressed the V5-tagged WcNdk1 and WcNdk2 in HEK293T cells and analyzed their distribution by immunofluorescence microscopy. WcNdk1 was found exclusively in the cytoplasm and was absent from cell compartments such as nucleus or Golgi (Fig. 5 ). WcNdk2 showed two mutually exclusive distribution patterns. In some cells the V5 signal was found throughout the nucleus, while in others, it was localized to perinuclear-Golgi regions, with no nuclear signal (Fig. 5 ). A similar nuclear localization of WcNdk2 was also observed in transfected HeLa cells (Supplementary Figure S2 ), indicating that this targeting is not cell-type specific. The Ndk inhibitor, Azidothymidine (AZT), inhibits the growth of W. chondrophila Since W. chondrophila is currently genetically intractable, we employed chemicals to inhibit Ndk function in this pathogen. AZT (3′-azido-3′-deoxythymidine), also known as zidovudine, is a known Ndk inhibitor 14,32–36 . This pharmaceutical, primarily approved for the treatment of human immunodeficiency virus (HIV), inhibits viral reverse transcription through chain termination 37 . This compound is structurally a thymidine analogue, where the 3’-hydroxyl group on the deoxyribose sugar is replaced by an azido group (N 3 ) (Fig. 6 A). The inhibition of the Ndk from Aspergillus flavus by AZT is documented, where AZT forms a strong hydrogen bond with key active site residues of Ndk (Arg-104, His-117 and Asp-120) and inhibits its enzymatic activity 14 . Since these three residues are highly conserved in Ndks of diverse organisms, including WcNdk1 and WcNdk2, we hypothesized that AZT could also serve as an effective inhibitor of Ndks in W. chondrophila (Fig. 2 A). To test this hypothesis, we treated both uninfected and W. chondrophila -infected McCoy cells with increasing concentrations of AZT following infection and measured cell death resulting from bacterial proliferation using propidium iodide (PI) staining. In the absence of AZT or at low concentrations (1 µg/ml), W. chondrophila -infected cultures exhibited a sharp increase in cell mortality over time, reaching maximal levels by 144 hpi. Treatment with AZT at concentrations above 25 µg/ml significantly suppressed infection-induced cell death. A delay in cell mortality was observed with the intermediate concentration of 5 µg/ml (Fig. 6 B, right panel). In contrast, in uninfected cells, cell lysis remained comparable across all AZT concentrations, showing no significant difference from the no-AZT control. Therefore, the slight increase in cell death observed over time in untreated cultures is due to normal cell aging and turnover, and not to AZT treatment (Fig. 6 B, left panel). These results indicate that AZT did not exhibit significant adverse effects on mammalian host cells and provided a protective effect against W. chondrophila -induced cell death in a dose-dependent manner. Only early AZT treatment prevents W. chondrophila –mediated host cell death To define the temporal window in which AZT exhibits its inhibitory effect on W. chondrophila growth, W. chondrophila -infected McCoy cell monolayers were treated with 25 µg/ml AZT at 0, 3, 8, 24, 32, or 48 hpi and the propidium iodide (PI) uptake was monitored at 24, 48, 72, and 144 hpi (Fig. 6 C). When AZT was added at 0, 3, or 8 hpi, cell mortality remained minimal (approximately 25% at 144 hpi), comparable to uninfected controls, indicating complete protection against bacterial-induced cell death. In contrast, administering AZT at 24 hpi or later failed to prevent cell death, resulting in maximal mortality, comparable to the no-AZT control. This indicates that once RB replication and inclusion expansion are established, AZT cannot reverse the course of infection. AZT induced the production of aberrant bodies (ABs) in W. chondrophila To investigate the cellular-level effects of AZT treatment on W. chondrophila infection, we performed confocal microscopy on W. chondrophila -infected McCoy cells treated with 25 µg/ml AZT at 0 hpi. In untreated cultures, W. chondrophila underwent normal intracellular development, leading to host cell lysis at the end of infection. At 8 hpi, W. chondrophila EBs were observed attached to the host cell surface and internalized in both AZT-treated and untreated cultures indicating that AZT does not interfere with bacterial attachment or entry (Fig. 6 D). By 24 hpi, inclusions had formed in both conditions. However, in AZT-treated cells, these inclusions failed to expand, and only a limited number of bacteria were detected within them. This suggests that although EB-to-RB differentiation and initial rounds of replication may occur, bacterial proliferation is subsequently arrested. In addition, the W. chondrophila RBs appeared slightly enlarged in AZT-treated cultures, consistent with the formation of aberrant bodies (ABs). These abnormal conditions persisted until 72 hpi, indicating a disruption of the normal developmental cycle likely resulting in bacterial persistence. Effect of AZT on W. chondrophila growth is probably correlated with WcNdk2 activity W. chondrophila encodes two ndk paralogs, organized in a single operon, with pabA positioned between them. To investigate which ndk copy mediates susceptibility to AZT, and whether this susceptibility is linked to the presence of the pabA gene, we selected representative Chlamydiota species with different ndk operon configurations and compared their response to AZT treatment. The selected species include C. trachomatis , which carries a single ctndk gene and lacks pabA ; Simkania negevensis , which also has a single snndk gene but retains pabA elsewhere in the genome; Estrella lausannensis , which harbors a two-gene elndk1 – elndk2 operon without pabA ; and W. chondrophila , which contains the full wcndk1 – pabA – wcndk2 operon (Fig. 7 , bottom panel). To evaluate the effect of AZT on these species, McCoy cell monolayers were infected, treated with 25 µg/ml at 0 hpi and fixed at 48 hpi before immunostaining and observation under confocal microscopy. As shown in Fig. 7 C, trachomatis and S. negevensis exhibited normal intracellular development in presence of AZT, indicating no significant growth inhibition. In contrast, E. lausannensis , like W. chondrophila , displayed enlarged intracellular structures resembling aberrant bodies. This suggest that AZT inhibitory effect depends on the presence of ndk2 , regardless of the presence of ndk1 and pabA . DISCUSSION The diversity of the ndk copy number within members of the Chlamydiota reveals lineage-specific adaptations with possible metabolic and pathogenic consequences. While most Chlamydiota species possess a single ndk gene, a subset, including members of the Parachlamydiaceae , Waddliaceae , and Criblamydiaceae , harbor a second copy ( ndk2 ), likely arising from a gene duplication event. This duplication may have provided a selective advantage by providing increasing metabolism flexibility, that could, in part, account for the comparatively faster growth of W. chondrophila and E. lausannensis in mammalian cells relative to other members of the Chlamydiota such as Chlamydia spp. and S. negevensis . Such an expansion of metabolic capacities might also represent an adaptation facilitating persistence and replication within free-living protists, suggesting that these lineages have evolved mechanisms enabling survival across diverse host environments. This is in line with their larger genome size (> 2 Mb), expanded metabolic genes and their reduced dependence on host-derived metabolites. On the other side, the absence of the second copy of the ndk gene in Rhabdochlamydiaceae , Simkaniaceae and Chlamydiaceae coincides with their adaptations to more specific host niches and reduced metabolic pathways. The pabA gene is also absent in all members of the Chlamydiaceae family. In this family, enzymes from other biosynthetic pathways are recruited to meet their folate requirements 38 . The mechanisms driving the evolution of this operon in certain chlamydial families remains unknown. Further studies are needed to clarify the functional role of this operon and its evolutionary pathways in these families. Both W. chondrophila ndk paralogs retain the universally conserved HGSD active-site motif and surrounding hydrophobic residues essential for autophosphorylation and phosphotransferase activities. This conservation suggests that both Ndk1 and Ndk2, despite their apparent different subcellular localizations, exert their functions, at least partially, through phosphorylation mechanisms. The temporal expression patterns of wcndk1 and wcndk2 provide insights into their potential biological roles and regulation during the W. chondrophila developmental cycle. The organization of wcndk1 and wcndk2 within a single operon explains their synchronized expression profiles. The apparent mid-cycle surge in Ndk protein at 24 hpi coincides with the maximal bacterial replication, an increased demand for NTPs, and increased host-manipulation activities that are essential for intracellular growth. Despite sharing conserved kinase domains, our study showed significant differences in subcellular localization of WcNdk1 and WcNdk2. This divergence suggests functional specializations and may indicate that the two proteins utilize divergent cellular trafficking mechanisms during W. chondrophila infection. In the C. trachomatis expression system, WcNdk1 was confined to the inclusion. This restricted distribution suggests that WcNdk1 may primarily serve bacterial intracellular functions within the inclusion. However, a previous study provided evidence for the secretion of WcNdk1 into the host cell cytosol 29 . It is, therefore, possible that WcNdk1 is secreted transiently or at levels insufficient to be detected via immunofluorescence. In contrast, WcNdk2 exhibited nuclear localization in C. trachomatis and HEK293T expression systems. This nuclear localization is consistent with previous studies on other pathogens, where Ndk was shown to bind host DNA and regulate gene expression through DNA cleavage 17,18 . Such putative nuclear activity suggests a potential role for WcNdk2 in reprogramming host transcriptional responses to favor bacterial survival or pathogenicity. It would be of great interest to identify specific host genes targeted by WcNdk2, as this could uncover previously unrecognized mechanisms by which W. chondrophila manipulates host cell function and could provide broader insights into its pathogenesis. When expressed in HEK293T cells, WcNdk2 localized not only to the nucleus but also to the perinuclear-Golgi region. Bacterial Ndks, such as Porphyromonas gingivalis Ndk, are found in the perinuclear area of the host cells. This localization has been linked to the modulation of host purinergic signaling via P2X₇ receptors 28 , implying that the perinuclear localization of WcNdk2 could be a biologically relevant phenomenon rather than nonspecific aggregation. Unlike the perinuclear accumulation of bacterial Ndks, Golgi localization represents a novel observation not previously reported for any bacterial species. One plausible explanation for an association of WcNdk2 with the Golgi is that, after translocation into the host cytosol, WcNdk2 enters the classical ER–Golgi secretory pathway and is packaged into Golgi-derived vesicles for extracellular release to hydrolyze host extracellular ATP (eATP) and subvert purinergic signaling. Another possible explanation for the Golgi recruitment of WcNdk2 is that it might play a role in modulating vesicle trafficking between the Golgi and the bacterial inclusion. Although W. chondrophila does not redirect sphingomyelin transport from the Golgi to its inclusion 39 , it may still influence other aspects of vesicular trafficking, altering vesicle formation or fusion processes, to benefit bacterial survival or replication. AZT is a well-established inhibitor of Ndk 14,32–36 . In our study, AZT treatment of W. chondrophila -infected cells protected them from bacteria-induced cell death and bacterial growth was arrested in the replication phase. Our comparative data across Chlamydiota species suggest that WcNdk2 is a potential target for AZT. WcNdk2 localizes to host cell compartments when expressed in heterologous systems and this positioning may increase its exposure to AZT. Thus, the growth arrest caused by AZT may result from disruption of Ndk2-dependent pathogenic mechanisms required for successful host-cell manipulation. Our results also showed that AZT must be applied before 24 hpi, to fully prevent W. chondrophila –induced cytotoxicity. This early phase of infection coincides with maximum RB replication, inclusion expansion, and active vesicular nutrient trafficking from the Golgi and ER 40 . In contrast, WcNdk1, which predominantly localizes within the bacteria, and is likely involved in maintaining bacterial nucleotide pools, appears less susceptible to AZT, possibly due to limited drug access to the inclusion compartment. These findings collectively support a model in which the inhibition of host-targeted, secreted WcNdk2, rather than the inclusion-confined WcNdk1, plays the key role in AZT’s antimicrobial activity against W. chondrophila . Based on these observations, we propose a functional model for the two W. chondrophila Ndks, as illustrated in Fig. 8. Together, these findings highlight the multifunctional nature of Ndk proteins and shed light on the distinct roles of W. chondrophila Ndks. However, the lack of genetic manipulation tools in W. chondrophila currently impedes direct investigation of Ndk function via gene deletion or mutagenesis. Although localization studies using heterologous expression systems suggest that WcNdk2 may interact with host cells, the possibility of overexpression artifacts or mis-localization cannot be entirely ruled out. The subcellular localizations of WcNdk2 are based on data obtained in heterologous expression systems. These localizations could not be directly observed in W. chondrophila infected cells, likely due to transient secretion and/or secretion of low amounts precluding the detection of the protein, as it is often the case for secreted effectors. Additionally, although AZT is a well-established Ndk inhibitor with no other known bacterial targets, direct in vitro evidence confirming WcNdk2 as the AZT target is still missing. In vitro ATPase assays are essential to validate this interaction. In the absence of such studies the proposed functions remain speculative. The advancement of genetic tools would be essential to validate the proposed roles of WcNdk1 and WcNdk2 in W. chondrophila metabolism and host interaction. CONCLUSION Given its diverse activities, Ndk has attracted interest across multiple disciplines, including microbiology, cell biology, and drug development. In this study, we introduced chlamydial Ndk as a multifunctional protein with roles extending beyond nucleotide metabolism, highlighting its potential involvement in host–pathogen interactions. These findings not only enhance our understanding of Ndk biology in the Chlamydiota phylum but also point to Ndk2 as a potential therapeutic target, opening new avenues for dissecting host manipulation strategies in obligate intracellular bacteria. MATERIALS AND METHODS Phylogenetic and structural analysis Species-based and ndk gene phylogenies were retrieved from the Chlamydia Database 41 . Trees were trimmed using FigTree (v1.4.4) to retain only relevant Chlamydiota species. The Ndk protein sequences across different taxa were retrieved from the National Center for Biotechnology Information (NCBI). Multiple sequence alignment of Ndk proteins across eukaryotes and prokaryotes was performed using the ClustalW tool in UGENE v1.30.0. The corresponding similarity heat map (percentage identity, excluding gap) was also generated on UGENE. Alignment visualization was carried out with EPSript https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi. The 3D structure of the protein was reconstructed using Phyre2 42 and visualized using Jmol v.16.2.15. Cell culture and W. chondrophila infection McCoy (murine fibroblast cells; ATCC CRL-1696, purchased from ATCC), HEK293T (ATCC CRL-11268, USA, obtained as a gift from Dr. Thierry Roger, Lausanne University Hospital) or HeLa cells (human cervical adenocarcinoma epithelial cells; ATCC CCL-2, a gift from Dr. Thierry Roger) were maintained at 37°C in 5% CO 2 in DMEM GlutaMAX (Thermo Fisher Scientific, USA), supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, USA). W. chondrophila (ATCC VR-1471) was propagated in Acanthamoeba castellanii (ATCC 30010) at 25°C in T-25 flasks containing 6 ml of peptone–yeast–extract–glucose broth. At the time of infection, lysed W. chondrophila -infected amoebae were filtered through a 5-µm syringe filter to remove amoebal debris. The bacterial solution was then used to infect the host cells at a dilution which was optimum for infection (MOI 0.1–1). To synchronize the infection, the infected cells were centrifuged at 1790 g for 10 minutes. They were then incubated at 37°C in 5% CO 2 for 15 minutes. Following incubation, theinoculum was then replaced with fresh medium. Purification of recombinant His-tagged WcNdk1 and WcNdk2 for antiserum production The E. coli strain BL21 containing pET28a- wcndk1-6xHis or pET28a- wcndk2-6xHis was grown to an OD 600 of 0.5. The culture was induced using 1 mM Isopropyl β-D-thiogalactopyranoside (IPTG, Applichem, Germany) and incubated at 37°C for 4 hours to express the recombinant protein. Bacterial pellets were resuspended in native lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, and 10 mM imidazole, pH 8). The resuspended cells were lysed using a combination of methods: three cycles of freeze (ethanol-dry ice bath) and thaw (at 37°C), followed by chemical lysis with 1 mg/ml lysozyme (AppliChem, Germany) and sonication. The recombinant protein was purified under native condition using Ni-NTA agarose beads (Qiagen, Germany) according to the manufacturer’s instructions. Briefly, bacterial lysates were incubated with Ni-NTA resin for 2 hours at 4 °C with gentle rotation. The protein-bound resin was then loaded onto poly-prep chromatography columns (Bio-Rad, USA), washed with wash buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0), and eluted with elution buffer containing 250 mM imidazole. The purified protein was dialyzed using Slide-A-Lyzer Dialysis Cassette, 2,000 MWCO (Thermo Scientific, USA) overnight against PBS to remove imidazole. The concentration of the protein was determined using Bradford’s reagent with BSA (Bio-Rad, USA) as a standard. Following protein purification, rabbit polyclonal antisera were produced using immunization services offered by Eurogentec SA (Seraing, Belgium). Western Blot McCoy cells were seeded at a density of 1 x 10 6 per T-25 flask one day before infection and infected with W. chondrophila as described above. Infected cells were harvested by scraping at the specified hpi and a fraction of the culture was saved for extraction and quantification of the genomic DNA (see below). The cell suspension was pelleted at 1790 g for 10 minutes, then washed twice with PBS, and finally resuspended in 0.5 ml of 1x Laemmli sample buffer (Bio-Rad, USA). An equal volume of each lysate was resolved on 12% SDS-PAGE precast gels (Bio-Rad, USA) and transferred onto an Amersham Protran nitrocellulose membrane (Cytiva, USA). The membrane was blocked in saturation buffer (10 mM Tris-Base, 150 mM NaCl, and 0.05% Tween 20) containing 5% nonfat dried milk (AppliChem, Germany) for 2 hours at room temperature (RT). Blots were probed overnight at 4°C with antibodies against WcNdk1 and WcNdk2 diluted in saturation buffer with 0.5% non-fat dried milk. After three washes with saturation buffer containing 0.5% non-fat dried milk, blots were incubated with secondary antibodies (horseradish peroxidase-conjugated anti-rabbit IgG; Promega, USA, or anti-mouse IgG; Bio-Rad, USA) for 1 hour at Room temperature and processed using the Amersham ECL detection system (Cytiva, USA). Western blot bands intensities were quantified using EvolutionCapt edge software (Vilber, France) and normalized to the corresponding bacterial genome copy number. Graphs were generated using GraphPad v. 10.4.1. Uncropped images of all blots are shown in the Supplementary Figure S3. Extraction of genomic DNA and quantification of W. chondrophila genome copy number Genomic DNA from collected samples was extracted using the Wizard SV Genomic DNA purification system (Promega, USA) according to the manufacturer’s instructions. The extracted DNA served as a template for qPCR with iTaq Universal Probes Supermix (Bio-Rad, USA) to quantify the W. chondrophila bacterial population. The 16S copy numbers were determined using a standard curve, which was generated from serial dilutions of a plasmid containing one copy of the 16S rRNA gene. The primer sequences 8 are listed in supplementary Table S1. RNA extraction and cDNA synthesis W. chondrophila- infected McCoy cell monolayers or uninfected control cells were harvested at indicated hpi by scraping and centrifugation (5000 g, 10 minutes). Following centrifugation cells were lysed directly in TRIzol Reagent (Invitrogen, USA). Total RNA was extracted by chloroform separation and recovery of the aqueous phase (12,000 g, 15 min, 4 °C). RNA was precipitated with isopropanol, washed with 75 % ethanol, air-dried, and resuspended in RNase‑free water. To remove genomic DNA, samples were treated with RNase‑free DNase I (Invitrogen, USA) following the manufacturer’s instructions. cDNA was synthesized using the GoScript Reverse Transcription System (Promega, USA) with random primers. Reverse transcription was performed at 42°C for 60 minutes, followed by enzyme inactivation at 70°C for 15 minutes. All cDNAs were diluted 1:5 prior to qPCR. RT-qPCR The qPCR reactions were set up in a final volume of 20 μl containing 4 μl of cDNA template, the appropriate concentration of each primer, and 1X iTaq Universal SYBR Green Supermix (Bio-Rad, USA). All samples were run in duplicates, and no-template controls were included for each primer pair to assess non-specific amplification. The fluorescent reporter signal was normalized against the internal reference dye (ROX) signal. qPCR was carried out on QuantStudio 3 Real-Time PCR System (Applied Biosystems, USA) using the following thermal program. A single cycle of DNA polymerase activation for 3 min at 95 °C, followed by 45 amplification cycles of 15 s at 95 °C (denaturing step) and 1 min at 60 °C (annealing and extension step). Gene expression data were normalized to W. chondrophila 16 rRNA gene, which served as the internal reference gene. Quantitative data from qRT-PCR experiments were collected from at least three independent replicates. The primers used for RT-qPCR are listed in supplementary Table S1. Application of AZT on W. chondrophila culture and cell death assessment McCoy cells were seeded at a density of 1 × 10 4 cells per well in 96-well plates (Corning, USA). Cells were infected with W. chondrophila or left uninfected as a control. To monitor cell death, Propidium Iodide (PI, 7 µg/mL, Sigma-Aldrich, Germany) was added to the growth medium. AZT was purchased from TOCRIS (Cat. No.:4150) and was added to the wells at concentrations ranging from 0 to 250 µg/mL in six technical replicates per condition. Depending on the experimental design, AZT was added at the time of infection (0 hpi) or at later time points (3, 8, 24, 32, and 48 hpi). PI fluorescence was measured at specified time points post-infection using a FLUOstar Omega plate reader (BMG LABTECH, Germany; excitation: 540 nm; emission: 640 nm). To define 100% cell death, 0.1% Triton X-100 was added to control wells prior to PI reading. Antibodies, immunofluorescence assay, and confocal microscopy Mouse anti-V5 antibody were purchased from Thermo Fisher Scientific (USA). Goat anti- Chlamydia trachomatis major outer membrane protein (MOMP) antibodies were obtained from Lifespan Bioscience (LS-C55983, USA). Polyclonal antibodies against W. chondrophila , E. lausannensis , and S. negevensis were homemade. Antibodies against GM130 (a cis-Golgi marker) were obtained from BD Biosciences (USA). Secondary anti-mouse, and anti-rabbit or anti-goat antibodies conjugated to Alexa Fluor 488 or 594, as well as Texas Red–conjugated concanavalin A, were purchased from AppliChem (Germany). McCoy cells were cultured on glass coverslips placed in 24-well-plates and infected with various bacterial species. At the indicated time points, cells were fixed with ice-cold methanol or 4% paraformaldehyde (PFA) for 5 minutes at 4°C, followed by three washes with phosphate-buffered saline (PBS). Fixed cells were permeabilized and blocked for 30 minutes using a blocking solution containing 0.1% saponin, 10% fetal bovine serum (FBS), and 0.04% sodium azide in PBS. Blocked cells were incubated with primary antibodies for 2 hours at RT (anti-V5 diluted 1:5000, anti-MOMP diluted 1:500 and anti GM130 diluted 1:300). After three washing steps with blocking solution, cells were incubated for 1 hour with Alexa Fluor 488- or 598-conjugated secondary antibodies (1:1000 dilution) to label bacteria. DAPI (4′,6-diamidino-2-phenylindole; Thermo Fisher Scientific, USA) (1:3000 dilution) and Concanavalin A (1:50 dilution) were used to stain nuclei and carbohydrates, respectively. Cells were then washed three times with PBS and briefly rinsed with purified water before mounting. Coverslips were mounted on glass slides with Mowiol 4-88 (Sigma-Aldrich, USA) and stored in the dark until imaging using Zeiss (LSM 900, Germany) confocal laser scanning microscope. pGL2 and pBOMBL plasmid construction The genes wcndk1 and wcndk2 were amplified from W. chondrophila genomic DNA using primers with 5′ overhangs compatible with the pGL2 (a kind gift from the Scott Hefty laboratory, University of Kansas) or pBOMBL (generously provided by Scot Ouellette laboratory, University of Nebraska Medical Center) backbone. All PCR products included a C-terminal V5 epitope tag to facilitate detection. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Germany). The pBOMBL vector was linearized with EagI and KpnI (NEB, USA). The native C. trachomatis –derived pGL2 vector (11,786 bp; β-lactamase selection marker) was linearized by Age I digestion (NEB, USA) and recovered from agarose gels with the QIAquick Gel Extraction Kit (Qiagen, Germany). Purified inserts and linearized vectors were assembled via In-fusion cloning using 5X In-fusion Snap assembly Master Mix (Takara Bio, Japan). Assemblies were incubated at 50 °C for 15 min. Transformations were performed using E. coli dam⁻ dcm⁻ cells. Positive clones were screened by colony PCR, and successful insertions were confirmed by Sanger sequencing using both vector- and insert-specific primers. C. trachomatis transformation Transformation of C. trachomatis was performed with minor modifications to previously described methods 43 .1 x 10 6 McCoy cells were seeded in 6-well plates and cultured overnight. 2.5 x 10 6 plasmid-free C. trachomatis serovar L2 (EBs), were resuspended in 300µl Tris-CaCl 2 buffer (10 mM Tris, 50 mM CaCl2, pH 7.4), and incubated with 2 µg of sequence-verified plasmid DNA for 30 minutes at room temperature. 1 mL Hank’s balanced salt solution (HBSS; Gibco, Thermo Fisher Scientific, USA) was then added to each reaction. This mixture was added to McCoy cells in a 6-well plate after removing the medium. The infection was carried out by centrifugation at 400 × g for 15 minutes at room temperature and incubation at 37°C for 15 minutes. Then, the inoculum was removed, and cells were incubated with 2 mL of DMEM medium supplemented with 10% FBS for 8 hours at 37°C and 5% CO₂. After this incubation, the medium was replaced with DMEM containing 10% FBS, 1 µg/mL cycloheximide (Sigma-Aldrich, Germany), and penicillin G (0.6 mg/mL; Sigma-Aldrich, Germany) or spectinomycin (50 µg/mL; Sigma-Aldrich, Germany) to select for transformed bacteria. Cells were passaged every 48 hours, and the development of fluorescent inclusions were monitored until they were clearly observed and established. The transformed C. trachomatis was harvested and titrated by IFU assay and stored at -80°C. For experiments requiring induction, 50 ng/ml anhydrotetracycline (aTc, Sigma-Aldrich, Germany) was added at the time of infection, whereas, in control conditions, the inducer was excluded. Gateway cloning for mammalian expression in pDEST47 For generation of pDEST47 expression plasmids, attB-flanked primers were used to amplify the C-terminal v5-tagged wcndk1 and wcndk2 . Entry clones were generated by BP recombination using the Gateway BP Clonase II Enzyme Mix (Thermo Fisher Scientific, USA). The resulting pDONR201- ndk-v5 entry clones were selected on Kanamycin (AppliChem, Germany) plates. Destination vectors were generated via LR recombination of the entry clones with pDEST47 using the Gateway LR Clonase II Enzyme Mix (Thermo Fisher Scientific, USA) following the supplier’s instructions. Final constructs were transformed and validated as described above, with plasmid DNA prepared from overnight cultures using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific, USA). Transient transfection of HEK293T and HeLa cells Glass coverslips placed in 24-well plates were coated with poly-L-lysine (100 µg/mL; Sigma-Aldrich, Germany) for 30 min at room temperature, washed three times with PBS, and air-dried for 3 h. HEK293T cells were seeded on coated coverslips at 2 × 10⁵ cells per well and incubated overnight at 37 °C in 5 % CO₂. HeLa cells were seeded on uncoated coverslips at 2.5 × 10⁵ cells per well and incubated under the same conditions. Twenty-four hours post-seeding, cells were transfected with pDEST47 expression plasmids using the Lipofectamine 3000 Transfection Kit (Thermo Fisher Scientific, USA). according to the manufacturer’s instructions. Briefly, for each well, 1 µg plasmid DNA was diluted in 25 µL serum-free DMEM containing 1 µL P3000 reagent. Separately, 0.75 µL Lipofectamine 3000 reagent was diluted in 25 µL serum-free DMEM. The two mixtures were combined, incubated for 15 min at room temperature, and 50 µL of the transfection complex was added dropwise per well. Cells were fixed 24 hours post-transfection with PFA 4% for downstream immunofluorescence analysis as described above. Declarations COMPETING INTERESTS The authors declare no competing interests. AUTHOR CONTRIBUTIONS G.G.S.: Conceptualization, Methodology, Investigation, Formal analysis, Visualization, Writing – original draft. C.K.B.: Investigation, Validation, Resources, Writing – review & editing. S.E.A.: Validation, Writing – review & editing. G.G.: Supervision, Conceptualization, Project administration, Funding acquisition, Writing – review & editing. DATA AVAILABILITY Data supporting the findings of this study are available from the first author (Giti Ghazi-Soltani) upon reasonable request. FUNDING This work was supported by the Swiss National Science Foundation (SNSF) [Grant No. 197768]. References Dilbeck PM, Evermann JF, Crawford TB, et al. Isolation of a previously undescribed rickettsia from an aborted bovine fetus. J Clin Microbiol. 1990;28(4):814-816. Henning K, Schares G, Granzow H, et al. Neospora caninum and Waddlia chondrophila strain 2032/99 in a septic stillborn calf. Veterinary Microbiology. 2002;85(3):285-292. doi:10.1016/S0378-1135(01)00510-7 Dilbeck-Robertson P, McAllister MM, Bradway D, Evermann JF. 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The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015;10(6):845-858. doi:10.1038/nprot.2015.053 Mueller KE, Wolf K, Fields KA. Chlamydia trachomatis transformation and allelic exchange mutagenesis. Curr Protoc Microbiol. 2017;45:11A.3.1-11A.3.15. doi:10.1002/cpmc.31 Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterialGhaziSoltani2025revised.pdf 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. 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1","display":"","copyAsset":false,"role":"figure","size":86625,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogeny of \u003cem\u003eChlamydiota \u003c/em\u003ephylum \u0026nbsp;\u0026nbsp;and the \u003cem\u003endk\u003c/em\u003e gene operon \u0026nbsp;\u0026nbsp;evolution. (A) Species phylogeny based on 32 single-copy orthologous genes. \u0026nbsp;\u0026nbsp;(B) Simplified representation of the species phylogeny highlighting major \u0026nbsp;\u0026nbsp;clades. (C) Phylogeny based on the \u003cem\u003endk\u003c/em\u003e gene, showing its evolutionary \u0026nbsp;\u0026nbsp;relationship among \u003cem\u003eChlamydial\u003c/em\u003e species.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8206315/v1/b6801330e466de0cdb8e56bd.jpg"},{"id":98635791,"identity":"95f8fcd9-7442-4bcd-903b-1ad1e98ddff5","added_by":"auto","created_at":"2025-12-19 17:26:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":165325,"visible":true,"origin":"","legend":"\u003cp\u003eNdk sequence and structure conservation. (A) Multiple sequence alignment of Ndk proteins across eukaryotes and prokaryotes. Conserved residues are highlighted in red with the highly conserved H residue, essential for catalytic activity, marked with an asterisk. Af: \u003cem\u003eAspergillus flavus\u003c/em\u003e, An: \u003cem\u003eA. niger\u003c/em\u003e, Hs: \u003cem\u003eHomo sapiens\u003c/em\u003e, Mt: \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e, Ec: \u003cem\u003eEscherichia coli, \u003c/em\u003eCt: \u003cem\u003eC. trachomatis\u003c/em\u003e, Sn: \u003cem\u003eSimkania negevensis\u003c/em\u003e, Rp: \u003cem\u003eRhabdochlamydia porcellionis, \u003c/em\u003eWc: \u003cem\u003eW. chondrophila\u003c/em\u003e, El: \u003cem\u003eEstrella lausannensis, \u003c/em\u003eCs: \u003cem\u003eCriblamydia sequanensis,\u003c/em\u003e Pa:\u003cem\u003e Parachlamydia acanthamoebae, \u003c/em\u003eNsp: \u003cem\u003eNeochlamydia sp.\u003c/em\u003e, Pae: \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, Ho: \u003cem\u003eHalofilum ochraceum\u003c/em\u003e, Lp: \u003cem\u003eLegionella pneumophila\u003c/em\u003e (B) Amino acid sequence similarity matrix comparing Ndk proteins from \u003cem\u003eW. chondrophila\u003c/em\u003e(WcNdk1 and WcNdk2), \u003cem\u003eChlamydia trachomatis\u003c/em\u003e (CtNdk), \u003cem\u003eEscherichia coli\u003c/em\u003e(EcNdk), \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e (MtNdk), and human Ndk (HsNdk). (C) Predicted 3D structure of WcNdk1, illustrating its conserved α/β fold characteristic of the Ndk family. 3D structure reconstructed using Phyre2 and visualized using Jmol v.16.2.15. The H177, highlighted in red, is a critical histidine residue and positioned within the active site.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8206315/v1/fe2ed5447a84eda2d0e36d82.jpg"},{"id":98635614,"identity":"a5e1c647-9e5b-4d2c-bbef-ae7461826535","added_by":"auto","created_at":"2025-12-19 17:26:22","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":21100,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal \u0026nbsp;\u0026nbsp;expression analysis of \u003cem\u003endk\u003c/em\u003e paralogs in \u003cem\u003eW. chondrophila\u003c/em\u003e during \u0026nbsp;\u0026nbsp;infection.\u003cbr\u003e\n \u0026nbsp;(A) Quantitative RT-PCR analysis of \u003cem\u003endk1\u003c/em\u003e and \u003cem\u003endk2\u003c/em\u003e \u0026nbsp;transcript levels at different hours post-infection (hpi), normalized to 16S \u0026nbsp;\u0026nbsp;rRNA. Transcript abundance is expressed relative to the 24 hpi time point, \u0026nbsp;\u0026nbsp;which was set as the reference (1.0). (B) Relative protein levels of WcNdk1 \u0026nbsp;\u0026nbsp;and WcNdk2 quantified by Western blot, normalized to bacterial genome copy \u0026nbsp;\u0026nbsp;number. Protein expression is shown relative to 24 hpi, set as 100%. (C) \u0026nbsp;\u0026nbsp;Representative Western blot images showing WcNdk1 and WcNdk2 expression \u0026nbsp;\u0026nbsp;across the infection time course. Data are shown as mean ± SD of 3 \u0026nbsp;\u0026nbsp;independent experiments.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8206315/v1/f7e6a24ecfe2b2f620362abd.jpg"},{"id":98635634,"identity":"6599717b-3ce2-466f-baf7-dd1b890cf5de","added_by":"auto","created_at":"2025-12-19 17:26:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":61062,"visible":true,"origin":"","legend":"\u003cp\u003eSubcellular localization of WcNdk1 and WcNdk2 overexpressed in \u003cem\u003eC. trachomatis. C. trachomatis\u003c/em\u003e was transformed with plasmids expressing either WcNdk1 or WcNdk2 tagged with a V5 epitope. Infected McCoy cells were fixed at 24 hpi. GFP (green) indicates transformed \u003cem\u003eC. trachomatis\u003c/em\u003e, anti-V5 (red) detects WcNdk proteins, and DAPI (blue) stains host cell nuclei and bacterial DNA. White arrows point to V5 signal within host nuclei.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8206315/v1/77a045162ea689d7e2bfc7ba.jpg"},{"id":98635425,"identity":"47ce68d4-13cd-408a-bf79-433ce2bf7572","added_by":"auto","created_at":"2025-12-19 17:26:13","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":102591,"visible":true,"origin":"","legend":"\u003cp\u003eSubcellular \u0026nbsp;\u0026nbsp;distribution of\u003cem\u003e \u003c/em\u003eWcNdk1 and WcNdk2 in HEK293T. HEK293T cells were \u0026nbsp;\u0026nbsp;transiently transfected with C-terminal V5-tagged WcNdk1 and WcNdk2 and \u0026nbsp;\u0026nbsp;stained with the Golgi marker GM130 (green), anti-V5 antibody (red), and DNA \u0026nbsp;\u0026nbsp;(DAPI, blue). High-magnification insets highlight the co-localization of \u0026nbsp;\u0026nbsp;WcNdk2 (red) with the Golgi (green) or the perinuclear region.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8206315/v1/7d5dce44318cbbd766e02a4b.jpg"},{"id":98635513,"identity":"7e032738-960e-46ba-b7bb-9152946f121e","added_by":"auto","created_at":"2025-12-19 17:26:15","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":75198,"visible":true,"origin":"","legend":"\u003cp\u003eAZT inhibits\u003cem\u003e W. chondrophila\u003c/em\u003e growth in infected host cells.\u003cem\u003e \u003c/em\u003e(A) Chemical structure of azidothymidine (AZT). (B) AZT treatment of uninfected host cells, serving as a negative control (left panel) and treatment of infected host cells at concentrations ≥25 μg/mL (right panel). (C) Effect of AZT addition on \u003cem\u003eW. chondrophila\u003c/em\u003e-infected cultures at various time points after infection. (D) Confocal microscopy of \u003cem\u003eW. chondrophila\u003c/em\u003e-infected cells treated or not treated with AZT. In untreated conditions, \u003cem\u003eW. chondrophila\u003c/em\u003e formed typical intracellular inclusions, whereas AZT treatment resulted in enlarged, aberrant structures. Bacteria are labeled using anti-\u003cem\u003eW. chondrophila \u003c/em\u003eantibody (green); host cell nuclei are stained with DAPI (blue); and the host structures are marked with Concanavalin A (red).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8206315/v1/f3548470b5739b4124a1c2ae.jpg"},{"id":98634763,"identity":"6c9d74ed-8f67-4e20-a5bb-3d6c93747515","added_by":"auto","created_at":"2025-12-19 17:25:52","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":168038,"visible":true,"origin":"","legend":"\u003cp\u003eAZT \u0026nbsp;\u0026nbsp;sensitivity is restricted to\u003cem\u003e Chlamydiota \u003c/em\u003especies encoding \u003cem\u003endk2\u003c/em\u003e. \u0026nbsp;\u0026nbsp;Representative confocal micrographs of McCoy cells infected with \u003cem\u003eC. \u0026nbsp;\u0026nbsp;trachomatis\u003c/em\u003e, \u003cem\u003eS. negevensis\u003c/em\u003e, \u003cem\u003eW. chondrophila\u003c/em\u003e or \u003cem\u003eE. \u0026nbsp;\u0026nbsp;lausannensis\u003c/em\u003e, treated or not treated with 25 μg/ml AZT. The infected \u0026nbsp;\u0026nbsp;cells were fixed and stained 48 hpi. The lower panel shows the \u003cem\u003endk\u003c/em\u003e \u0026nbsp;operon arrangement: \u003cem\u003eC. trachomatis\u003c/em\u003e and \u003cem\u003eS. negevensis\u003c/em\u003e, each carry \u0026nbsp;\u0026nbsp;a single\u003cem\u003e ndk\u003c/em\u003e gene. \u003cem\u003eS. negevensis \u003c/em\u003eretains \u003cem\u003epabA\u003c/em\u003e elsewhere \u0026nbsp;\u0026nbsp;in the genome. \u003cem\u003eW. chondrophila\u003c/em\u003e carries the two \u003cem\u003ewcndk\u003c/em\u003e genes with \u0026nbsp;\u0026nbsp;a \u003cem\u003epabA\u003c/em\u003e in the middle. \u003cem\u003eE. lausannensis\u003c/em\u003e encodes a two-gene \u003cem\u003eelndk1–elndk2\u003c/em\u003e \u0026nbsp;operon but lacks \u003cem\u003epabA\u003c/em\u003e. \u003cem\u003eBacteria \u0026nbsp;\u0026nbsp;are\u003c/em\u003e stained with species-specific antibodies. \u0026nbsp;Host cytoplasm is labeled with Concanavalin A (red), and nuclei are stained \u0026nbsp;\u0026nbsp;with DAPI (blue). \u003cem\u003ectndk\u003c/em\u003e: \u003cem\u003eC. trachomatis ndk1\u003c/em\u003e, \u003cem\u003esnndk1\u003c/em\u003e:\u003cem\u003e \u0026nbsp;S. negevensis ndk1\u003c/em\u003e, \u003cem\u003eelndk1\u003c/em\u003e: \u003cem\u003e\u0026nbsp;E. lausannensis ndk1\u003c/em\u003e, \u003cem\u003eelndk2\u003c/em\u003e:\u003cem\u003e \u0026nbsp;E. lausannensis ndk2.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8206315/v1/fc15da8396427e701a539403.jpg"},{"id":98635244,"identity":"25784d84-50d9-4ae0-8787-d68375d8f10d","added_by":"auto","created_at":"2025-12-19 17:26:10","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":100143,"visible":true,"origin":"","legend":"\u003cp\u003eProposed model for the functions of \u0026nbsp;\u0026nbsp;WcNdk1 and WcNdk2 in\u003cem\u003e W. chondrophila. \u003c/em\u003eThis schematic illustrates the \u0026nbsp;\u0026nbsp;distinct subcellular localizations and putative roles of WcNdk1 (green) and \u0026nbsp;\u0026nbsp;WcNdk2 (red), during infection of a host cell. A: WcNdk1 is predominantly \u0026nbsp;\u0026nbsp;retained within the bacterial inclusion, where it sustains basic nucleotide \u0026nbsp;\u0026nbsp;metabolism and “housekeeping” functions. B: A fraction of WcNdk1 is secreted \u0026nbsp;\u0026nbsp;across the inclusion membrane into the host cytosol, however, the specific \u0026nbsp;\u0026nbsp;functions of cytosolic WcNdk1 remain undefined. C: WcNdk2 is observed \u0026nbsp;\u0026nbsp;accumulating within the host nucleus. Its import into the nucleus could occur \u0026nbsp;\u0026nbsp;via one or more of the following mechanisms: a deacetylation event allowing \u0026nbsp;\u0026nbsp;passive nuclear import; free diffusion through nuclear pores owing to its \u0026nbsp;\u0026nbsp;small molecular size; or interaction with host proteins that escort WcNdk2 \u0026nbsp;\u0026nbsp;into the nucleus. Inside host nucleus, WcNdk2 could bind directly to DNA \u0026nbsp;\u0026nbsp;leading to up‐ or downregulation of host genes involved in immunity or \u0026nbsp;\u0026nbsp;apoptosis. The precise gene targets remain unknown. D: WcNdk2 traffics \u0026nbsp;\u0026nbsp;through Golgi/ER‐derived vesicles and perinuclear region. This localization \u0026nbsp;\u0026nbsp;may be related to the packaging of WcNdk2 into vesicles destined for \u0026nbsp;\u0026nbsp;secretion to subvert host purinergic signaling through hydrolysis of eATP \u0026nbsp;\u0026nbsp;into ADP. E: The association of WcNdk2 with the Golgi apparatus \u0026nbsp;may imply \u0026nbsp;\u0026nbsp;an involvement in vesicle trafficking and acquisition of host-derived \u0026nbsp;\u0026nbsp;metabolites necessary for expansion of the inclusion and bacterial survival. Illustration generated using Adobe Illustrator 2024 \u0026nbsp;\u0026nbsp;(Adobe Inc., USA).\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8206315/v1/2da67cea13224b833ff62a6f.jpg"},{"id":104975159,"identity":"dec2b34c-5fe9-4ae2-a0fc-69f66980f37c","added_by":"auto","created_at":"2026-03-19 11:57:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1869614,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8206315/v1/4292c5ab-bc83-455e-909b-1bc69ccb8e79.pdf"},{"id":98635056,"identity":"93f4c0ea-892b-425f-bd18-30be6eb14787","added_by":"auto","created_at":"2025-12-19 17:26:01","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":734821,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialGhaziSoltani2025revised.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8206315/v1/b5d017e99fbbe708f84aaa8f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functional insights into nucleoside diphosphate kinases encoded by two ndk paralogs in Waddlia chondrophila","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe \u003cem\u003eChlamydiota\u003c/em\u003e phylum encompasses several families of obligate intracellular bacteria, including six main families: \u003cem\u003eChlamydiaceae\u003c/em\u003e, \u003cem\u003eParachlamydiaceae\u003c/em\u003e, \u003cem\u003eWaddliaceae\u003c/em\u003e, \u003cem\u003eSimkaniaceae\u003c/em\u003e, \u003cem\u003eRhabdochlamydiaceae\u003c/em\u003e, and \u003cem\u003eCriblamydiaceae\u003c/em\u003e. Members of these families share a biphasic developmental cycle, which reflects their adaptation to intracellular environments and reliance on host cell machinery for survival.\u003c/p\u003e \u003cp\u003e \u003cem\u003eW. chondrophila\u003c/em\u003e belongs to the family \u003cem\u003eWaddliaceae\u003c/em\u003e and is known as an abortigenic agent in ruminants. It was first isolated from an aborted bovine fetus\u003csup\u003e1,2\u003c/sup\u003e and serological studies showed a strong association between anti-\u003cem\u003eWaddlia\u003c/em\u003e antibodies and bovine abortion\u003csup\u003e3\u003c/sup\u003e. In humans, \u003cem\u003eW. chondrophila\u003c/em\u003e seropositivity is significantly associated with adverse pregnancy outcomes\u003csup\u003e4\u0026ndash;6\u003c/sup\u003e. This pathogen was also detected in respiratory tract samples of patients with pneumonia and children with bronchiolitis\u003csup\u003e7,8\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eW. chondrophila\u003c/em\u003e developmental cycle is biphasic and is divided into three major phases\u003csup\u003e9,10\u003c/sup\u003e. In the early phase (0\u0026ndash;8 hours post-infection, hpi) elementary bodies (EBs) enter in host cells and differentiate into reticulate bodies (RBs). This phase is followed by a proliferation phase (8\u0026ndash;24 hpi) during which the number of RBs increases exponentially through binary fission. In the late phase (24\u0026ndash;48 hpi), RBs revert into EBs and lysis of the host cell releases infectious progeny. Under environmental or antibiotic stress, the developmental cycle is arrested leading to an enlarged, non-dividing RB-like form called Aberrant Body (AB)\u003csup\u003e11\u003c/sup\u003e. While \u003cem\u003eW. chondrophila\u003c/em\u003e significantly impacts both animal and human health, the specific molecular and genomic pathways, particularly those involving developmental and regulatory proteins, remain largely unexplored. Functional characterization of proteins, such as nucleoside diphosphate kinase (Ndk), could help us to better understand the biology and pathogenicity of this pathogen and its adaptation inside host cells.\u003c/p\u003e \u003cp\u003eNdk is a highly conserved enzyme that plays a central role in maintaining nucleotide pools within both bacterial and eukaryotic cells. The maintenance of intracellular NTP pools is vital for the survival and proliferation of all living organisms. Ndk regulates nucleotide pools through its autophosphorylation and phosphotransferase activities. This ensures that NTP and dNTP levels remain balanced for optimal cellular function.\u003c/p\u003e \u003cp\u003eBeyond its housekeeping functions, Ndk is implicated in various cellular processes, including cell differentiation and development in eukaryotes and prokaryotes\u003csup\u003e12\u0026ndash;15\u003c/sup\u003e. Additionally, Ndk is involved in the regulation of gene expression\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e. Due to its ability to bind ATP, Ndk is involved in inhibiting extracellular ATP (eATP) and disrupting purinergic signaling in immune and inflammatory responses\u003csup\u003e19\u0026ndash;21\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe previously reported transcriptomic data for \u003cem\u003eW. chondrophila\u003c/em\u003e comparing genes expression in RBs at 24 hpi and EBs at 72 hpi\u003csup\u003e22\u003c/sup\u003e. In that dataset, both \u003cem\u003endk\u003c/em\u003e genes were differentially expressed between the two developmental stages, each showing a fold change of 0.3 in EBs compared with RBs. Despite the well-established functions of Ndk across various organisms, its specific function in the \u003cem\u003eChlamydiota\u003c/em\u003e remains uncharacterized. Based on these observations, we hypothesized that this enzyme could play an important role in the development and pathogenesis of \u003cem\u003eW. chondrophila\u003c/em\u003e. In this study, we characterize the two Ndk proteins in this organism to better understand their potential role in the bacterium\u0026rsquo;s development. This study provides an overall view of Ndk proteins in the \u003cem\u003eChlamydiota\u003c/em\u003e phylum and offers functional insights into the Ndks proteins of \u003cem\u003eW. chondrophila\u003c/em\u003e. Our results pave the way for more detailed research once genetic tools become available for \u003cem\u003eW. chondrophila\u003c/em\u003e.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eEvolution of the\u003c/b\u003e \u003cb\u003endk\u003c/b\u003e \u003cb\u003eoperon in the\u003c/b\u003e \u003cb\u003eChlamydiota\u003c/b\u003e \u003cb\u003ephylum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSimilar to other organisms, all bacteria of the \u003cem\u003eChlamydiota\u003c/em\u003e phylum possess at least one copy of the \u003cem\u003endk\u003c/em\u003e gene. A second copy (\u003cem\u003endk2\u003c/em\u003e) is present in members of the \u003cem\u003eParachlamydiaceae\u003c/em\u003e, \u003cem\u003eWaddliaceae\u003c/em\u003e, and \u003cem\u003eCriblamydiaceae\u003c/em\u003e families (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Interestingly, \u003cem\u003endk2\u003c/em\u003e forms an operon with the ancestral \u003cem\u003endk1\u003c/em\u003e gene. In most species, this operon is further associated with \u003cem\u003epabA\u003c/em\u003e, which encodes para-aminobenzoate synthase and is involved in the folate biosynthesis pathway. Comparison of gene-based and species-based phylogenetic trees (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C) suggests that the duplication of \u003cem\u003endk2\u003c/em\u003e most likely originated from a single gene duplication event in the common ancestor of the \u003cem\u003eParachlamydiaceae\u003c/em\u003e, \u003cem\u003eWaddliaceae\u003c/em\u003e, and \u003cem\u003eCriblamydiaceae\u003c/em\u003e. However, a scenario of ancestral duplication with subsequent loss in \u003cem\u003eSimkaniaceae\u003c/em\u003e and \u003cem\u003eChlamydiaceae\u003c/em\u003e cannot be excluded, although it is less parsimonious.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHigh sequence and structural conservation of Ndk across species\u003c/h2\u003e \u003cp\u003eThe multiple sequence alignment shows a high degree of sequence conservation of Ndk proteins from various bacterial and eukaryotic species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Specifically, the His-Gly-Ser-Asp (HGSD) motif, which is the enzyme\u0026rsquo;s active site\u003csup\u003e23\u003c/sup\u003e, is well conserved across all aligned sequences. The highly conserved histidine residue inside this motif transiently receives the phosphate group during autophosphorylation reaction and is, therefore, critical for the catalytic function of Ndk. In addition to the HGSD motif, other conserved residues are observed in regions associated with nucleotide binding and structural stability, many of which are enriched in hydrophobic amino acids. These residues likely contribute to the proper folding and enzymatic efficiency of Ndk.\u003c/p\u003e \u003cp\u003e \u003cem\u003eW. chondrophila\u003c/em\u003e contains two copies of \u003cem\u003endk\u003c/em\u003e: \u003cem\u003ewcndk1 (\u003c/em\u003elocus \u003cem\u003ewcw_1543\u003c/em\u003e) and \u003cem\u003ewcndk2\u003c/em\u003e (locus \u003cem\u003ewcw_1545\u003c/em\u003e), with a sequence similarity of 54% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The most striking difference between the two \u003cem\u003eW. chondrophila ndk\u003c/em\u003e paralogs is the presence of a predicted signal peptide of 17 amino acids in WcNdk2, which is absent in WcNdk1.\u003c/p\u003e \u003cp\u003eThe sequence similarity matrix of \u003cem\u003eW. chondrophila\u003c/em\u003e Ndk proteins, WcNdk1 and WcNdk2, and their counterparts from other bacterial and eukaryotic species shows differing degrees of conservation as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. WcNdk1 shares the highest sequence identity with \u003cem\u003eE. coli\u003c/em\u003e Ndk (EcNdk) and \u003cem\u003eChlamydia trachomatis\u003c/em\u003e Ndk (CtNdk) (67% and 66%, respectively). In contrast, WcNdk2 exhibits slightly lower sequence similarity across species, which may indicate functional divergence of the two Ndks following gene duplication. Interestingly, both \u003cem\u003eW. chondrophila\u003c/em\u003e Ndk homologs display relatively high sequence identity (~\u0026thinsp;50%) with human Ndk (HsNdk), which suggests the conservation of core functional domains across vast evolutionary distance. Sequence similarity matrix of Ndk proteins across different species is shown in supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eWcNdk1 has 143 residues forming a polypeptide chain and adopting a very similar fold to Ndk proteins from other origins, including human Ndk\u003csup\u003e24,25\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). One Ndk unit has α/β domains comprising a four-stranded antiparallel β-sheet and two connecting α-helices. This high degree of sequence and structural conservation between eukaryotes and pathogens may enable them to interfere with host cellular processes by mimicking host Ndk.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression profile of\u003c/b\u003e \u003cb\u003eW. chondrophila\u003c/b\u003e \u003cb\u003eNdks during infection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTemporal expression levels of genes and proteins across different developmental stages may help us understand their stage-specific roles and regulatory mechanisms. Since each of these stages has unique biological features, we can indirectly infer a protein\u0026rsquo;s function by mapping both transcript and protein levels across these developmental stages.\u003c/p\u003e \u003cp\u003eWe quantified the expression profiles of the \u003cem\u003eW. chondrophila ndks\u003c/em\u003e, at both the transcript and protein level during McCoy cell infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u003cem\u003ewcndk1\u003c/em\u003e and \u003cem\u003ewcndk2\u003c/em\u003e transcripts were highly abundant at early time points (3\u0026ndash;8 hpi) as revealed by RT-qPCR, then the mRNA level declined sharply to a minimum at 32 hpi, with a moderate surge toward the end of the cycle (48 hpi) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In parallel, protein expression profiles of WcNdk1and WcNdk2 were analyzed using antibodies specifically raised against each protein. No WcNdk1 or WcNdk2 protein could be detected by Western blot of the \u003cem\u003eW. chondrophila\u003c/em\u003e-infected cells at 3 to 8 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Both proteins became detectable by 24 hpi by Western blot and reached their highest signal intensity at this time point after normalization to bacterial number, followed by a drop to roughly 30% of that level by 48 hpi. To further assess protein expression at early stages, we performed immunofluorescence microscopy. In contrast to Western blot results, WcNdk1 and WcNdk2 signals were already detectable from 0 to 16 hpi, colocalizing with \u003cem\u003eW. chondrophila\u003c/em\u003e (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Together, these results suggests that both WcNdk1 and WcNdk2 are expressed throughout the developmental cycle, with an early transcriptional peak. While protein expression begins early, overall accumulation or detection sensitivity by Western blot peaks around 24 hpi, followed by a decline toward the end of the cycle.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSubcellular localization of WcNdk1 and WcNdk2 in\u003c/b\u003e \u003cb\u003eC. trachomatis\u003c/b\u003e \u003cb\u003eheterologous expression\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhile signal peptides are typically associated with protein secretion, previous studies have demonstrated that Ndk can be secreted in certain bacterial species despite lacking a canonical signal sequence\u003csup\u003e26\u0026ndash;28\u003c/sup\u003e. Moreover, the secretion of both WcNdk1 and WcNdk2 into the host cell cytoplasm was experimentally shown\u003csup\u003e29\u003c/sup\u003e, suggesting that WcNdk2 signal peptide may not contribute to secretion. The presence of a signal peptide on WcNdk2 suggests that this protein may be translocated to a specific subcellular compartment, giving it a biological role distinct from WcNdk1. To assess this possibility, we overexpressed a V5-tagged versions of WcNdk1 and WcNdk2 in \u003cem\u003eC. trachomatis\u003c/em\u003e using the inducible shuttle vector pGL2 and studied their subcellular localization by immunofluorescence and confocal microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In this heterologous expression assay, WcNdk1 was exclusively detected inside the inclusion, closely associated with \u003cem\u003eC. trachomatis\u003c/em\u003e, and no signal was observed in the host cell cytoplasm. Although this does not rule out secretion of WcNdk1, it suggests limited accumulation or detection outside the inclusion. In contrast, WcNdk2 was detected in both chlamydial inclusions and the host-cell nucleus, implying that it may be transported into the nucleus. To further investigate the nuclear localization of WcNdk2, we cloned the gene into the pBOMBL vector\u003csup\u003e30\u003c/sup\u003e and overexpressed the protein in \u003cem\u003eC. trachomatis\u003c/em\u003e. Upon infection of McCoy cells with the transformed bacteria, inclusions appeared smaller compared to controls. Due to these growth defects, reliable localization of WcNdk2 could not be realized in this system. This vector-associated toxicity likely reflects bacterial stress caused by excessive or mis-regulated expression of WcNdk2, similar to the overexpression-associated toxicity reported for type III effectors in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003csup\u003e31\u003c/sup\u003e. Consistently, WcNdk2 expression was also not detected in the \u003cem\u003eYersinia enterocolitica\u003c/em\u003e type III secretion assay, suggesting poor tolerance or instability of the protein in this system.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSubcellular localization of WcNdk1 and WcNdk2 in transfected HEK293T cells\u003c/h3\u003e\n\u003cp\u003eTo further confirm the observed subcellular localization of WcNdk1 and WcNdk2 in another heterologous system, we transiently expressed the V5-tagged WcNdk1 and WcNdk2 in HEK293T cells and analyzed their distribution by immunofluorescence microscopy. WcNdk1 was found exclusively in the cytoplasm and was absent from cell compartments such as nucleus or Golgi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). WcNdk2 showed two mutually exclusive distribution patterns. In some cells the V5 signal was found throughout the nucleus, while in others, it was localized to perinuclear-Golgi regions, with no nuclear signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). A similar nuclear localization of WcNdk2 was also observed in transfected HeLa cells (Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), indicating that this targeting is not cell-type specific.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe Ndk inhibitor, Azidothymidine (AZT), inhibits the growth of\u003c/b\u003e \u003cb\u003eW. chondrophila\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSince \u003cem\u003eW. chondrophila\u003c/em\u003e is currently genetically intractable, we employed chemicals to inhibit Ndk function in this pathogen. AZT (3\u0026prime;-azido-3\u0026prime;-deoxythymidine), also known as zidovudine, is a known Ndk inhibitor\u003csup\u003e14,32\u0026ndash;36\u003c/sup\u003e. This pharmaceutical, primarily approved for the treatment of human immunodeficiency virus (HIV), inhibits viral reverse transcription through chain termination\u003csup\u003e37\u003c/sup\u003e. This compound is structurally a thymidine analogue, where the 3\u0026rsquo;-hydroxyl group on the deoxyribose sugar is replaced by an azido group (N\u003csub\u003e3\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThe inhibition of the Ndk from \u003cem\u003eAspergillus flavus\u003c/em\u003e by AZT is documented, where AZT forms a strong hydrogen bond with key active site residues of Ndk (Arg-104, His-117 and Asp-120) and inhibits its enzymatic activity\u003csup\u003e14\u003c/sup\u003e. Since these three residues are highly conserved in Ndks of diverse organisms, including WcNdk1 and WcNdk2, we hypothesized that AZT could also serve as an effective inhibitor of Ndks in \u003cem\u003eW. chondrophila\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eTo test this hypothesis, we treated both uninfected and \u003cem\u003eW. chondrophila\u003c/em\u003e-infected McCoy cells with increasing concentrations of AZT following infection and measured cell death resulting from bacterial proliferation using propidium iodide (PI) staining. In the absence of AZT or at low concentrations (1 \u0026micro;g/ml), \u003cem\u003eW. chondrophila\u003c/em\u003e-infected cultures exhibited a sharp increase in cell mortality over time, reaching maximal levels by 144 hpi. Treatment with AZT at concentrations above 25 \u0026micro;g/ml significantly suppressed infection-induced cell death. A delay in cell mortality was observed with the intermediate concentration of 5 \u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, right panel). In contrast, in uninfected cells, cell lysis remained comparable across all AZT concentrations, showing no significant difference from the no-AZT control. Therefore, the slight increase in cell death observed over time in untreated cultures is due to normal cell aging and turnover, and not to AZT treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, left panel). These results indicate that AZT did not exhibit significant adverse effects on mammalian host cells and provided a protective effect against \u003cem\u003eW. chondrophila\u003c/em\u003e-induced cell death in a dose-dependent manner.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOnly early AZT treatment prevents\u003c/b\u003e \u003cb\u003eW. chondrophila\u003c/b\u003e\u003cb\u003e\u0026ndash;mediated host cell death\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo define the temporal window in which AZT exhibits its inhibitory effect on \u003cem\u003eW. chondrophila\u003c/em\u003e growth, \u003cem\u003eW. chondrophila\u003c/em\u003e-infected McCoy cell monolayers were treated with 25 \u0026micro;g/ml AZT at 0, 3, 8, 24, 32, or 48 hpi and the propidium iodide (PI) uptake was monitored at 24, 48, 72, and 144 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). When AZT was added at 0, 3, or 8 hpi, cell mortality remained minimal (approximately 25% at 144 hpi), comparable to uninfected controls, indicating complete protection against bacterial-induced cell death. In contrast, administering AZT at 24 hpi or later failed to prevent cell death, resulting in maximal mortality, comparable to the no-AZT control. This indicates that once RB replication and inclusion expansion are established, AZT cannot reverse the course of infection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAZT induced the production of aberrant bodies (ABs) in\u003c/b\u003e \u003cb\u003eW. chondrophila\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the cellular-level effects of AZT treatment on \u003cem\u003eW. chondrophila\u003c/em\u003e infection, we performed confocal microscopy on \u003cem\u003eW. chondrophila\u003c/em\u003e-infected McCoy cells treated with 25 \u0026micro;g/ml AZT at 0 hpi. In untreated cultures, \u003cem\u003eW. chondrophila\u003c/em\u003e underwent normal intracellular development, leading to host cell lysis at the end of infection. At 8 hpi, \u003cem\u003eW. chondrophila\u003c/em\u003e EBs were observed attached to the host cell surface and internalized in both AZT-treated and untreated cultures indicating that AZT does not interfere with bacterial attachment or entry (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). By 24 hpi, inclusions had formed in both conditions. However, in AZT-treated cells, these inclusions failed to expand, and only a limited number of bacteria were detected within them. This suggests that although EB-to-RB differentiation and initial rounds of replication may occur, bacterial proliferation is subsequently arrested. In addition, the \u003cem\u003eW. chondrophila\u003c/em\u003e RBs appeared slightly enlarged in AZT-treated cultures, consistent with the formation of aberrant bodies (ABs). These abnormal conditions persisted until 72 hpi, indicating a disruption of the normal developmental cycle likely resulting in bacterial persistence.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of AZT on\u003c/b\u003e \u003cb\u003eW. chondrophila\u003c/b\u003e \u003cb\u003egrowth is probably correlated with WcNdk2 activity\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eW. chondrophila\u003c/em\u003e encodes two \u003cem\u003endk\u003c/em\u003e paralogs, organized in a single operon, with \u003cem\u003epabA\u003c/em\u003e positioned between them. To investigate which \u003cem\u003endk\u003c/em\u003e copy mediates susceptibility to AZT, and whether this susceptibility is linked to the presence of the \u003cem\u003epabA\u003c/em\u003e gene, we selected representative \u003cem\u003eChlamydiota\u003c/em\u003e species with different \u003cem\u003endk\u003c/em\u003e operon configurations and compared their response to AZT treatment. The selected species include \u003cem\u003eC. trachomatis\u003c/em\u003e, which carries a single \u003cem\u003ectndk\u003c/em\u003e gene and lacks \u003cem\u003epabA\u003c/em\u003e; \u003cem\u003eSimkania negevensis\u003c/em\u003e, which also has a single \u003cem\u003esnndk\u003c/em\u003e gene but retains \u003cem\u003epabA\u003c/em\u003e elsewhere in the genome; \u003cem\u003eEstrella lausannensis\u003c/em\u003e, which harbors a two-gene \u003cem\u003eelndk1\u003c/em\u003e\u0026ndash;\u003cem\u003eelndk2\u003c/em\u003e operon without \u003cem\u003epabA\u003c/em\u003e; and \u003cem\u003eW. chondrophila\u003c/em\u003e, which contains the full \u003cem\u003ewcndk1\u003c/em\u003e\u0026ndash;\u003cem\u003epabA\u003c/em\u003e\u0026ndash;\u003cem\u003ewcndk2\u003c/em\u003e operon (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, bottom panel). To evaluate the effect of AZT on these species, McCoy cell monolayers were infected, treated with 25 \u0026micro;g/ml at 0 hpi and fixed at 48 hpi before immunostaining and observation under confocal microscopy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, \u003cem\u003etrachomatis\u003c/em\u003e and \u003cem\u003eS. negevensis\u003c/em\u003e exhibited normal intracellular development in presence of AZT, indicating no significant growth inhibition. In contrast, \u003cem\u003eE. lausannensis\u003c/em\u003e, like \u003cem\u003eW. chondrophila\u003c/em\u003e, displayed enlarged intracellular structures resembling aberrant bodies. This suggest that AZT inhibitory effect depends on the presence of \u003cem\u003endk2\u003c/em\u003e, regardless of the presence of \u003cem\u003endk1\u003c/em\u003e and \u003cem\u003epabA\u003c/em\u003e.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe diversity of the \u003cem\u003endk\u003c/em\u003e copy number within members of the \u003cem\u003eChlamydiota\u003c/em\u003e reveals lineage-specific adaptations with possible metabolic and pathogenic consequences. While most \u003cem\u003eChlamydiota\u003c/em\u003e species possess a single \u003cem\u003endk\u003c/em\u003e gene, a subset, including members of the \u003cem\u003eParachlamydiaceae\u003c/em\u003e, \u003cem\u003eWaddliaceae\u003c/em\u003e, and \u003cem\u003eCriblamydiaceae\u003c/em\u003e, harbor a second copy (\u003cem\u003endk2\u003c/em\u003e), likely arising from a gene duplication event. This duplication may have provided a selective advantage by providing increasing metabolism flexibility, that could, in part, account for the comparatively faster growth of \u003cem\u003eW. chondrophila\u003c/em\u003e and \u003cem\u003eE. lausannensis\u003c/em\u003e in mammalian cells relative to other members of the \u003cem\u003eChlamydiota\u003c/em\u003e such as \u003cem\u003eChlamydia\u003c/em\u003e spp. and \u003cem\u003eS. negevensis\u003c/em\u003e. Such an expansion of metabolic capacities might also represent an adaptation facilitating persistence and replication within free-living protists, suggesting that these lineages have evolved mechanisms enabling survival across diverse host environments. This is in line with their larger genome size (\u0026gt;\u0026thinsp;2 Mb), expanded metabolic genes and their reduced dependence on host-derived metabolites. On the other side, the absence of the second copy of the \u003cem\u003endk\u003c/em\u003e gene in \u003cem\u003eRhabdochlamydiaceae\u003c/em\u003e, \u003cem\u003eSimkaniaceae\u003c/em\u003e and \u003cem\u003eChlamydiaceae\u003c/em\u003e coincides with their adaptations to more specific host niches and reduced metabolic pathways. The \u003cem\u003epabA\u003c/em\u003e gene is also absent in all members of the \u003cem\u003eChlamydiaceae\u003c/em\u003e family. In this family, enzymes from other biosynthetic pathways are recruited to meet their folate requirements\u003csup\u003e38\u003c/sup\u003e. The mechanisms driving the evolution of this operon in certain chlamydial families remains unknown. Further studies are needed to clarify the functional role of this operon and its evolutionary pathways in these families.\u003c/p\u003e \u003cp\u003eBoth \u003cem\u003eW. chondrophila ndk\u003c/em\u003e paralogs retain the universally conserved HGSD active-site motif and surrounding hydrophobic residues essential for autophosphorylation and phosphotransferase activities. This conservation suggests that both Ndk1 and Ndk2, despite their apparent different subcellular localizations, exert their functions, at least partially, through phosphorylation mechanisms.\u003c/p\u003e \u003cp\u003eThe temporal expression patterns of \u003cem\u003ewcndk1\u003c/em\u003e and \u003cem\u003ewcndk2\u003c/em\u003e provide insights into their potential biological roles and regulation during the \u003cem\u003eW. chondrophila\u003c/em\u003e developmental cycle. The organization of \u003cem\u003ewcndk1\u003c/em\u003e and \u003cem\u003ewcndk2\u003c/em\u003e within a single operon explains their synchronized expression profiles. The apparent mid-cycle surge in Ndk protein at 24 hpi coincides with the maximal bacterial replication, an increased demand for NTPs, and increased host-manipulation activities that are essential for intracellular growth.\u003c/p\u003e \u003cp\u003eDespite sharing conserved kinase domains, our study showed significant differences in subcellular localization of WcNdk1 and WcNdk2. This divergence suggests functional specializations and may indicate that the two proteins utilize divergent cellular trafficking mechanisms during \u003cem\u003eW. chondrophila\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003eIn the \u003cem\u003eC. trachomatis\u003c/em\u003e expression system, WcNdk1 was confined to the inclusion. This restricted distribution suggests that WcNdk1 may primarily serve bacterial intracellular functions within the inclusion. However, a previous study provided evidence for the secretion of WcNdk1 into the host cell cytosol\u003csup\u003e29\u003c/sup\u003e. It is, therefore, possible that WcNdk1 is secreted transiently or at levels insufficient to be detected via immunofluorescence. In contrast, WcNdk2 exhibited nuclear localization in \u003cem\u003eC. trachomatis\u003c/em\u003e and HEK293T expression systems. This nuclear localization is consistent with previous studies on other pathogens, where Ndk was shown to bind host DNA and regulate gene expression through DNA cleavage\u003csup\u003e17,18\u003c/sup\u003e. Such putative nuclear activity suggests a potential role for WcNdk2 in reprogramming host transcriptional responses to favor bacterial survival or pathogenicity. It would be of great interest to identify specific host genes targeted by WcNdk2, as this could uncover previously unrecognized mechanisms by which \u003cem\u003eW. chondrophila\u003c/em\u003e manipulates host cell function and could provide broader insights into its pathogenesis.\u003c/p\u003e \u003cp\u003eWhen expressed in HEK293T cells, WcNdk2 localized not only to the nucleus but also to the perinuclear-Golgi region. Bacterial Ndks, such as \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e Ndk, are found in the perinuclear area of the host cells. This localization has been linked to the modulation of host purinergic signaling via P2X₇ receptors\u003csup\u003e28\u003c/sup\u003e, implying that the perinuclear localization of WcNdk2 could be a biologically relevant phenomenon rather than nonspecific aggregation. Unlike the perinuclear accumulation of bacterial Ndks, Golgi localization represents a novel observation not previously reported for any bacterial species. One plausible explanation for an association of WcNdk2 with the Golgi is that, after translocation into the host cytosol, WcNdk2 enters the classical ER\u0026ndash;Golgi secretory pathway and is packaged into Golgi-derived vesicles for extracellular release to hydrolyze host extracellular ATP (eATP) and subvert purinergic signaling. Another possible explanation for the Golgi recruitment of WcNdk2 is that it might play a role in modulating vesicle trafficking between the Golgi and the bacterial inclusion. Although \u003cem\u003eW. chondrophila\u003c/em\u003e does not redirect sphingomyelin transport from the Golgi to its inclusion\u003csup\u003e39\u003c/sup\u003e, it may still influence other aspects of vesicular trafficking, altering vesicle formation or fusion processes, to benefit bacterial survival or replication.\u003c/p\u003e \u003cp\u003eAZT is a well-established inhibitor of Ndk\u003csup\u003e14,32\u0026ndash;36\u003c/sup\u003e. In our study, AZT treatment of \u003cem\u003eW. chondrophila\u003c/em\u003e-infected cells protected them from bacteria-induced cell death and bacterial growth was arrested in the replication phase. Our comparative data across \u003cem\u003eChlamydiota\u003c/em\u003e species suggest that WcNdk2 is a potential target for AZT. WcNdk2 localizes to host cell compartments when expressed in heterologous systems and this positioning may increase its exposure to AZT. Thus, the growth arrest caused by AZT may result from disruption of Ndk2-dependent pathogenic mechanisms required for successful host-cell manipulation. Our results also showed that AZT must be applied before 24 hpi, to fully prevent \u003cem\u003eW. chondrophila\u003c/em\u003e\u0026ndash;induced cytotoxicity. This early phase of infection coincides with maximum RB replication, inclusion expansion, and active vesicular nutrient trafficking from the Golgi and ER\u003csup\u003e40\u003c/sup\u003e. In contrast, WcNdk1, which predominantly localizes within the bacteria, and is likely involved in maintaining bacterial nucleotide pools, appears less susceptible to AZT, possibly due to limited drug access to the inclusion compartment. These findings collectively support a model in which the inhibition of host-targeted, secreted WcNdk2, rather than the inclusion-confined WcNdk1, plays the key role in AZT\u0026rsquo;s antimicrobial activity against \u003cem\u003eW. chondrophila\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eBased on these observations, we propose a functional model for the two \u003cem\u003eW. chondrophila\u003c/em\u003e Ndks, as illustrated in Fig.\u0026nbsp;8. Together, these findings highlight the multifunctional nature of Ndk proteins and shed light on the distinct roles of \u003cem\u003eW. chondrophila\u003c/em\u003e Ndks. However, the lack of genetic manipulation tools in \u003cem\u003eW. chondrophila\u003c/em\u003e currently impedes direct investigation of Ndk function via gene deletion or mutagenesis. Although localization studies using heterologous expression systems suggest that WcNdk2 may interact with host cells, the possibility of overexpression artifacts or mis-localization cannot be entirely ruled out. The subcellular localizations of WcNdk2 are based on data obtained in heterologous expression systems. These localizations could not be directly observed in \u003cem\u003eW. chondrophila\u003c/em\u003e infected cells, likely due to transient secretion and/or secretion of low amounts precluding the detection of the protein, as it is often the case for secreted effectors. Additionally, although AZT is a well-established Ndk inhibitor with no other known bacterial targets, direct in vitro evidence confirming WcNdk2 as the AZT target is still missing. In vitro ATPase assays are essential to validate this interaction. In the absence of such studies the proposed functions remain speculative. The advancement of genetic tools would be essential to validate the proposed roles of WcNdk1 and WcNdk2 in \u003cem\u003eW. chondrophila\u003c/em\u003e metabolism and host interaction.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eGiven its diverse activities, Ndk has attracted interest across multiple disciplines, including microbiology, cell biology, and drug development. In this study, we introduced chlamydial Ndk as a multifunctional protein with roles extending beyond nucleotide metabolism, highlighting its potential involvement in host\u0026ndash;pathogen interactions. These findings not only enhance our understanding of Ndk biology in the \u003cem\u003eChlamydiota\u003c/em\u003e phylum but also point to Ndk2 as a potential therapeutic target, opening new avenues for dissecting host manipulation strategies in obligate intracellular bacteria.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003ePhylogenetic and structural analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecies-based and \u003cem\u003endk\u003c/em\u003e gene phylogenies were retrieved from the \u003cem\u003eChlamydia\u003c/em\u003e Database\u003csup\u003e41\u003c/sup\u003e. Trees were trimmed using FigTree (v1.4.4) to retain only relevant \u003cem\u003eChlamydiota\u003c/em\u003e species. The Ndk protein sequences across different taxa were retrieved from the National Center for Biotechnology Information (NCBI). Multiple sequence alignment of Ndk proteins across eukaryotes and prokaryotes was performed using the ClustalW tool in UGENE v1.30.0. \u0026nbsp;The corresponding similarity heat map (percentage identity, excluding gap) was also generated on UGENE. Alignment visualization was carried out with EPSript https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi. The 3D structure of the protein was reconstructed using Phyre2\u003csup\u003e42\u003c/sup\u003e and visualized using Jmol v.16.2.15.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and \u003cem\u003eW. chondrophila\u0026nbsp;\u003c/em\u003einfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMcCoy (murine fibroblast cells; ATCC CRL-1696, purchased from ATCC), HEK293T (ATCC CRL-11268, USA, obtained as a gift from Dr. Thierry Roger, Lausanne University Hospital) or HeLa cells (human cervical adenocarcinoma epithelial cells; ATCC CCL-2, a gift from Dr. Thierry Roger) were maintained at 37°C in 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ein DMEM GlutaMAX (Thermo Fisher Scientific, USA), supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, USA). \u003cem\u003eW. chondrophila\u003c/em\u003e (ATCC VR-1471)\u0026nbsp;was propagated in \u003cem\u003eAcanthamoeba castellanii\u003c/em\u003e (ATCC 30010) at 25°C in T-25\u0026nbsp;flasks containing 6 ml of peptone–yeast–extract–glucose broth. At the time of infection, lysed \u003cem\u003eW. chondrophila\u003c/em\u003e-infected amoebae were filtered through a 5-µm\u0026nbsp;syringe filter to remove amoebal debris. The bacterial solution was then used to infect the host cells at a dilution which was optimum for infection (MOI 0.1–1). To synchronize the infection, the infected cells were centrifuged at 1790 g for 10 minutes. They were then incubated at 37°C in 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003efor 15 minutes. Following incubation, theinoculum was then replaced with fresh medium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePurification of recombinant His-tagged WcNdk1 and WcNdk2 for antiserum production\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eE. coli\u003c/em\u003e strain BL21 containing pET28a-\u003cem\u003ewcndk1-6xHis\u0026nbsp;\u003c/em\u003eor pET28a-\u003cem\u003ewcndk2-6xHis\u0026nbsp;\u003c/em\u003ewas grown to an OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003eof 0.5. The culture was induced using 1 mM Isopropyl β-D-thiogalactopyranoside (IPTG,\u0026nbsp;Applichem, Germany) and incubated at 37°C for 4 hours to express the recombinant protein. Bacterial pellets were resuspended in native lysis buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 300 mM NaCl, and 10 mM imidazole, pH 8). The resuspended cells were lysed using a combination of methods: \u0026nbsp;three cycles of freeze (ethanol-dry ice bath) and thaw (at 37°C), followed by chemical lysis with 1 mg/ml lysozyme (AppliChem, Germany) and sonication. The recombinant protein was purified under native condition using Ni-NTA agarose beads (Qiagen, Germany) according to the manufacturer’s instructions. Briefly, bacterial lysates were incubated with Ni-NTA resin for 2 hours at 4 °C with gentle rotation. The protein-bound resin was then loaded onto poly-prep chromatography columns (Bio-Rad, USA), washed with wash buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0), and eluted with elution buffer containing 250 mM imidazole. The purified protein was dialyzed using Slide-A-Lyzer Dialysis Cassette, 2,000 MWCO (Thermo Scientific, USA) overnight against PBS to remove imidazole. The concentration of the protein was determined using Bradford’s reagent with BSA (Bio-Rad, USA) as a standard. Following protein purification, rabbit polyclonal antisera were produced using immunization services offered by Eurogentec SA (Seraing, Belgium).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMcCoy cells were seeded at a density of 1 x 10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003e per T-25 flask one day before infection and infected with \u003cem\u003eW. chondrophila\u003c/em\u003e as described above. Infected cells were harvested by scraping at the specified hpi and a fraction of the culture was saved for extraction and quantification of the genomic DNA (see below). The cell suspension was pelleted at 1790 g for 10 minutes, then washed twice with PBS, and finally resuspended in 0.5 ml of 1x Laemmli sample buffer (Bio-Rad, USA). An equal volume of each lysate was resolved on 12% SDS-PAGE precast gels (Bio-Rad, USA) and transferred onto an Amersham Protran nitrocellulose membrane (Cytiva, USA). The membrane was blocked in saturation buffer (10 mM Tris-Base, 150 mM NaCl, and 0.05% Tween 20) containing 5% nonfat dried milk (AppliChem, Germany) for 2 hours at room temperature (RT). Blots were probed overnight at 4°C with antibodies against WcNdk1 and WcNdk2 diluted in saturation buffer with 0.5% non-fat dried milk. After three washes with saturation buffer containing 0.5% non-fat dried milk, blots were incubated with secondary antibodies (horseradish peroxidase-conjugated anti-rabbit IgG; Promega, USA, or anti-mouse IgG; Bio-Rad, USA) for 1 hour at Room temperature and processed using the Amersham ECL detection system (Cytiva, USA). Western blot bands intensities were quantified using EvolutionCapt edge software (Vilber, France) and normalized to the corresponding bacterial genome copy number. Graphs were generated using GraphPad v. 10.4.1. Uncropped images of all blots are shown in the Supplementary Figure S3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction of genomic DNA and quantification of \u003cem\u003eW. chondrophila\u0026nbsp;\u003c/em\u003egenome copy number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA from collected samples was extracted using the Wizard SV Genomic DNA purification system (Promega, USA) according to the manufacturer’s instructions. The extracted DNA served as a template for qPCR with iTaq Universal Probes Supermix (Bio-Rad, USA) to quantify the \u003cem\u003eW. chondrophila\u003c/em\u003e bacterial population. The 16S copy numbers were determined using a standard curve, which was generated from serial dilutions of a plasmid containing one copy of the 16S rRNA gene. The primer sequences\u003csup\u003e8\u003c/sup\u003e are listed in supplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and cDNA synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eW. chondrophila-\u003c/em\u003einfected McCoy cell monolayers or uninfected control cells were harvested at indicated hpi by scraping and centrifugation (5000 g, 10 minutes). Following centrifugation cells were lysed directly in TRIzol Reagent (Invitrogen, USA). Total RNA was extracted by chloroform separation and recovery of the aqueous phase (12,000 g, 15 min, 4 °C). RNA was precipitated with isopropanol, washed with 75 % ethanol, air-dried, and resuspended in RNase‑free water. To remove genomic DNA, samples were treated with RNase‑free DNase I (Invitrogen, USA) following the manufacturer’s instructions. cDNA was synthesized using the GoScript Reverse Transcription System (Promega, USA) with random primers. Reverse transcription was performed at 42°C for 60 minutes, followed by enzyme inactivation at 70°C for 15 minutes. All cDNAs were diluted 1:5 prior to qPCR.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe qPCR reactions were set up in a final volume of 20\u0026nbsp;μl containing 4 μl of cDNA template, the appropriate concentration of each primer, and 1X iTaq Universal SYBR Green Supermix (Bio-Rad, USA). All samples were run in duplicates, and no-template controls were included for each primer pair to assess non-specific amplification. The fluorescent reporter signal was normalized against the internal reference dye (ROX) signal. qPCR was carried out on QuantStudio 3 Real-Time PCR System (Applied Biosystems, USA) using the following thermal program. A single cycle of DNA polymerase activation for 3 min at 95 °C, followed by 45 amplification cycles of 15 s at 95 °C (denaturing step) and 1 min at 60 °C (annealing and extension step). Gene expression data were normalized to \u003cem\u003eW. chondrophila\u003c/em\u003e 16 rRNA gene, which served as the internal reference gene. Quantitative data from qRT-PCR experiments were collected from at least three independent replicates. The primers used for RT-qPCR are listed in supplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApplication of AZT on \u003cem\u003eW. chondrophila\u0026nbsp;\u003c/em\u003eculture and cell death assessment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMcCoy cells were seeded at a density of 1 × 10\u003csup\u003e4\u003c/sup\u003e cells per well in 96-well plates (Corning, USA). Cells were infected with \u003cem\u003eW. chondrophila\u0026nbsp;\u003c/em\u003eor left uninfected as a control. To monitor cell death, Propidium Iodide (PI, 7\u0026nbsp;µg/mL, Sigma-Aldrich, Germany) was added to the growth medium. AZT was purchased from TOCRIS (Cat. No.:4150) and was added to the wells at concentrations ranging from 0 to 250 µg/mL in six technical replicates per condition. Depending on the experimental design, AZT was added at the time of infection (0 hpi) or at later time points (3, 8, 24, 32, and 48 hpi). PI fluorescence was measured at specified time points post-infection using a FLUOstar Omega plate reader (BMG LABTECH, Germany; excitation: 540 nm; emission: 640 nm). To define 100% cell death, 0.1% Triton X-100 was added to control wells prior to PI reading.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibodies, immunofluorescence assay, and confocal microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse anti-V5 antibody were purchased from Thermo Fisher Scientific (USA). Goat anti-\u003cem\u003eChlamydia trachomatis\u003c/em\u003e major outer membrane protein (MOMP) antibodies were obtained from Lifespan Bioscience (LS-C55983, USA). Polyclonal antibodies against \u003cem\u003eW. chondrophila\u003c/em\u003e, \u003cem\u003eE. lausannensis\u003c/em\u003e, and \u003cem\u003eS. negevensis\u003c/em\u003e were homemade. Antibodies against GM130 (a cis-Golgi marker) were obtained from BD Biosciences (USA). Secondary anti-mouse, and anti-rabbit or anti-goat antibodies conjugated to Alexa Fluor 488 or 594, as well as Texas Red–conjugated concanavalin A, were purchased from AppliChem (Germany).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMcCoy cells were cultured on glass coverslips placed in 24-well-plates and infected with various bacterial species. At the indicated time points, cells were fixed with ice-cold methanol or 4% paraformaldehyde (PFA) for 5 minutes at 4°C, followed by three washes with phosphate-buffered saline (PBS). Fixed cells were permeabilized and blocked for 30 minutes using a blocking solution containing 0.1% saponin,\u0026nbsp;10% fetal bovine serum (FBS), and 0.04% sodium azide in PBS. Blocked cells were incubated with primary antibodies for 2 hours at RT (anti-V5 diluted 1:5000, anti-MOMP diluted 1:500 and anti GM130 diluted 1:300). After three washing steps with blocking solution, cells were incubated for 1 hour with Alexa Fluor 488- or 598-conjugated secondary antibodies (1:1000 dilution) to label bacteria. DAPI (4′,6-diamidino-2-phenylindole; Thermo Fisher Scientific, USA) (1:3000 dilution) and Concanavalin A (1:50 dilution) were used to stain nuclei and carbohydrates, respectively. Cells were then washed three times with PBS and briefly rinsed with purified water before mounting. Coverslips were mounted on glass slides with Mowiol 4-88 (Sigma-Aldrich, USA)\u0026nbsp;and stored in the dark until imaging using Zeiss (LSM 900, Germany) confocal laser scanning microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003epGL2 and pBOMBL plasmid construction\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genes \u003cem\u003ewcndk1\u003c/em\u003e and \u003cem\u003ewcndk2\u003c/em\u003e were amplified from \u003cem\u003eW. chondrophila\u0026nbsp;\u003c/em\u003egenomic DNA using primers with 5′ overhangs compatible with the pGL2 (a kind gift from the Scott Hefty laboratory, University of Kansas) or pBOMBL (generously provided by Scot Ouellette laboratory, University of Nebraska Medical Center) backbone. All PCR products included a C-terminal V5 epitope tag to facilitate detection. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Germany). The pBOMBL vector was linearized with EagI and KpnI (NEB, USA). The native \u003cem\u003eC. trachomatis\u003c/em\u003e–derived pGL2 vector (11,786 bp; β-lactamase selection marker) was linearized by Age I digestion (NEB, USA) and recovered from agarose gels with the QIAquick Gel Extraction Kit (Qiagen, Germany). Purified inserts and linearized vectors were assembled via In-fusion cloning using 5X In-fusion Snap assembly Master Mix (Takara Bio, Japan). Assemblies were incubated at 50 °C for 15 min. Transformations were performed using \u003cem\u003eE. coli\u003c/em\u003e dam⁻ dcm⁻ cells. Positive clones were screened by colony PCR, and successful insertions were confirmed by Sanger sequencing using both vector- and insert-specific primers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. trachomatis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;transformation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransformation of \u003cem\u003eC. trachomatis\u003c/em\u003e was performed with minor modifications to previously described methods\u003csup\u003e43\u003c/sup\u003e.1 x 10\u003csup\u003e6\u003c/sup\u003e McCoy cells were seeded in 6-well plates and cultured overnight. 2.5 x 10\u003csup\u003e6\u003c/sup\u003e plasmid-free \u003cem\u003eC. trachomatis serovar L2\u0026nbsp;\u003c/em\u003e(EBs), were resuspended in 300µl Tris-CaCl\u003csub\u003e2\u003c/sub\u003e buffer (10 mM Tris, 50 mM CaCl2, pH 7.4), and incubated with 2 µg of sequence-verified plasmid DNA for 30 minutes at room temperature. 1 mL Hank’s balanced salt solution (HBSS; Gibco, Thermo Fisher Scientific, USA) was then added to each reaction. This mixture was added to McCoy cells in a 6-well plate after removing the medium. The infection was carried out by centrifugation at 400 × g for 15 minutes at room temperature and incubation at 37°C for 15 minutes. Then, the inoculum was removed, and cells were incubated with 2 mL of DMEM medium supplemented with 10% FBS for 8 hours at 37°C and 5% CO₂. After this incubation, the medium was replaced with DMEM containing 10% FBS, 1 µg/mL cycloheximide (Sigma-Aldrich, Germany), and penicillin G (0.6 mg/mL; Sigma-Aldrich, Germany) or spectinomycin (50 µg/mL;\u0026nbsp;Sigma-Aldrich, Germany) to select for transformed bacteria. Cells were passaged every 48 hours, and the development of fluorescent inclusions were monitored until they were clearly observed and established. The transformed \u003cem\u003eC. trachomatis\u003c/em\u003e was harvested and titrated by IFU assay and stored at -80°C. For experiments requiring induction, 50 ng/ml anhydrotetracycline (aTc, Sigma-Aldrich, Germany) was added at the time of infection, whereas, in control conditions, the inducer was excluded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGateway cloning for mammalian expression in pDEST47\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor generation of pDEST47 expression plasmids, \u003cstrong\u003eattB-flanked primers\u003c/strong\u003e were used to amplify the \u003cstrong\u003eC-terminal v5-tagged\u0026nbsp;\u003c/strong\u003e\u003cem\u003ewcndk1\u003c/em\u003e and \u003cem\u003ewcndk2\u003c/em\u003e. Entry clones were generated by BP recombination using the Gateway BP Clonase II Enzyme Mix (Thermo Fisher Scientific, USA). The resulting pDONR201-\u003cem\u003endk-v5\u003c/em\u003e entry clones were selected on Kanamycin (AppliChem, Germany) plates. Destination vectors were generated via LR recombination of the entry clones with pDEST47 using the Gateway LR Clonase II Enzyme Mix (Thermo Fisher Scientific, USA)\u0026nbsp;following the supplier’s instructions.\u0026nbsp;Final constructs were transformed and validated as described above, with plasmid DNA prepared from overnight cultures using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransient transfection of HEK293T and HeLa cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGlass coverslips placed in 24-well plates were coated with poly-L-lysine (100 µg/mL; Sigma-Aldrich, Germany) for 30 min at room temperature, washed three times with PBS, and air-dried for 3 h. HEK293T cells were seeded on coated coverslips at 2 × 10⁵ cells per well and incubated overnight at 37 °C in 5 % CO₂. HeLa cells were seeded on uncoated coverslips at 2.5 × 10⁵ cells per well and incubated under the same conditions. Twenty-four hours post-seeding, cells were transfected with pDEST47 expression plasmids using the Lipofectamine 3000 Transfection Kit (Thermo Fisher Scientific, USA). according to the manufacturer’s instructions. Briefly, for each well, 1 µg plasmid DNA was diluted in 25 µL serum-free DMEM containing 1 µL P3000 reagent. Separately, 0.75 µL Lipofectamine 3000 reagent was diluted in 25 µL serum-free DMEM. The two mixtures were combined, incubated for 15 min at room temperature, and 50 µL of the transfection complex was added dropwise per well. Cells were fixed 24 hours post-transfection with PFA 4% for downstream immunofluorescence analysis as described above.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.G.S.: Conceptualization, Methodology, Investigation, Formal analysis, Visualization, Writing \u0026ndash; original draft.\u003c/p\u003e\n\u003cp\u003eC.K.B.: Investigation, Validation, Resources, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eS.E.A.: Validation, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eG.G.: Supervision, Conceptualization, Project administration, Funding acquisition, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are available from the first author (Giti Ghazi-Soltani) upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Swiss National Science Foundation (SNSF) [Grant No. 197768].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDilbeck PM, Evermann JF, Crawford TB, et al. 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Promiscuous and Adaptable Enzymes Fill \u0026ldquo;Holes\u0026rdquo; in the Tetrahydrofolate Pathway in Chlamydia Species. mBio. 2014;5(4):e01378-14. doi:10.1128/mBio.01378-14\u003c/li\u003e\n\u003cli\u003eDille S, Kleinschnitz EM, Kontchou CW, N\u0026ouml;lke T, H\u0026auml;cker G. In contrast to Chlamydia trachomatis, Waddlia chondrophila grows in human cells without inhibiting apoptosis, fragmenting the Golgi apparatus, or diverting post-Golgi sphingomyelin transport. Infect Immun. 2015;83(8):3268-3280. doi:10.1128/IAI.00322-15\u003c/li\u003e\n\u003cli\u003eCroxatto A, Greub G. Early intracellular trafficking of Waddlia chondrophila in human macrophages. Microbiology (Reading). 2010;156(Pt 2):340-355. doi:10.1099/mic.0.034546-0\u003c/li\u003e\n\u003cli\u003ePillonel T, Tagini F, Bertelli C, Greub G. ChlamDB: a comparative genomics database of the phylum Chlamydiae and other members of the Planctomycetes-Verrucomicrobiae-Chlamydiae superphylum. Nucleic Acids Research. 2020;48(D1):D526-D534. doi:10.1093/nar/gkz924\u003c/li\u003e\n\u003cli\u003eKelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015;10(6):845-858. doi:10.1038/nprot.2015.053\u003c/li\u003e\n\u003cli\u003eMueller KE, Wolf K, Fields KA. Chlamydia trachomatis transformation and allelic exchange mutagenesis. Curr Protoc Microbiol. 2017;45:11A.3.1-11A.3.15. doi:10.1002/cpmc.31\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":"Waddlia chondrophila, Nucleoside diphosphate kinase (Ndk), AZT inhibition, Subcellular localization, Protein trafficking","lastPublishedDoi":"10.21203/rs.3.rs-8206315/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8206315/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe\u003cem\u003e Chlamydiota \u003c/em\u003ephylum consists of obligate intracellular bacteria, including well-known pathogens and emerging environmental species, with diverse host ranges and metabolic capabilities. Among these bacteria, the gene, which encodes nucleoside diphosphate kinase (\u003cem\u003endk\u003c/em\u003e), is present in variable copy numbers. While most chlamydial species carry a single copy of \u003cem\u003endk\u003c/em\u003e, some species have two copies. In \u003cem\u003eW. chondrophila\u003c/em\u003e, the two Ndk proteins encoded by \u003cem\u003endk\u003c/em\u003e paralogs retain conserved kinase motifs but differ in subcellular localization, suggesting divergent functional roles. According to localization studies performed inheterologous expression systems, \u003cem\u003eWcNdk1\u003c/em\u003eis confined to the inclusion and probably supports nucleotide metabolism, while \u003cem\u003eWcNdk2\u003c/em\u003e localizes to the host nucleus, perinuclear space, and Golgi apparatus, suggesting involvement in host interaction. Azidothymidine (AZT), a known Ndk inhibitor, impaired \u003cem\u003eW. chondrophila growth\u003c/em\u003e, potentially through inhibition of \u003cem\u003eWcNdk2\u003c/em\u003e. \u003cem\u003eHowever, the lack of genetic tools and the absence of in vitro enzymatic assays currently limit definitive functional conclusions. Our data suggest \u003c/em\u003epotential functions for Ndks in \u003cem\u003eW. chondrophila, \u003c/em\u003eproviding \u003cem\u003ea foundation for future studies on Ndk-mediated interactions between this pathogen and its host.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"Functional insights into nucleoside diphosphate kinases encoded by two ndk paralogs in Waddlia chondrophila","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-19 17:17:13","doi":"10.21203/rs.3.rs-8206315/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":"2dc316c8-e59d-4388-a443-0f5e481efa22","owner":[],"postedDate":"December 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59911018,"name":"Biological sciences/Biochemistry"},{"id":59911019,"name":"Biological sciences/Evolution"},{"id":59911020,"name":"Biological sciences/Microbiology"},{"id":59911021,"name":"Biological sciences/Molecular biology"},{"id":59911022,"name":"Biological sciences/Structural biology"}],"tags":[],"updatedAt":"2026-03-19T11:55:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-19 17:17:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8206315","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8206315","identity":"rs-8206315","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}