Biology subjects

Cox, J. V.

Publications and source records attributed to Cox, J. V..

5 recordsLinked to original sources

bioRxiv2026

Pkn1 kinase is a critical regulator of cell division in Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular, developmentally regulated pathogen with a reduced genome as a consequence of adapting to the host cell environment. Despite possessing only eight transcription factors, its developmental cycle is tightly regulated. Hence, we hypothesize that factors like post-transcriptional and post-translational modifications play significant roles in controlling developmental progression. Chlamydia encodes serine/threonine kinases, including an ortholog of Pkn1. In this study, we characterized the function of Pkn1 in chlamydial biology and revealed its role as a cell division regulator. We designed an anhydrotetracycline (aTc)-inducible construct encoding Pkn1 tagged with six histidines at the C-terminus (Pkn1_6xH). Wild-type Pkn1 localized primarily at the division septum whereas a kinase-null Pkn1 isoform localized in the cytosol. To further explore the association of Pkn1 with the divisome, we designed a plasmid encoding an aTc-inducible CRISPRi system targeting pkn1(pkn1KD). Intriguingly, decreased pkn1 transcript levels resulted in enlarged chlamydial cells with irregular coccoid shapes akin to penicillin-treated cells, indicating a block in cell division when pkn1 transcripts are reduced. These effects were complemented by expressing an additional wild-type copy, but not a kinase-null copy, of pkn1 during knockdown. Using a bacterial two hybrid system, we identified an interaction between Pkn1 and MreB, a cytoskeletal protein forming a ring at the septum in Chlamydia. We showed the co-localization of these proteins in Chlamydia and that inhibition of MreB function disrupts Pkn1 localization. Overall, our data indicate an essential function for Pkn1 activity in regulating cell division in Chlamydia.

microbiology↗

bioRxiv2024

FtsK Initiates the Assembly of a Unique Divisome Complex in the FtsZ-less Chlamydia trachomatis

Chlamydia trachomatis serovar L2 (Ct), an obligate intracellular bacterium that does not encode FtsZ, divides by a polarized budding process. In the absence of FtsZ, we show that FtsK, a chromosomal translocase, is critical for divisome assembly in Ct. Chlamydial FtsK forms discrete foci at the septum and at the base of the progenitor mother cell, and our data indicate that FtsK foci at the base of the mother cell mark the location of nascent divisome complexes that form at the site where a daughter cell will emerge in the next round of division. The divisome in Ct has a hybrid composition, containing elements of the divisome and elongasome from other bacteria, and FtsK is recruited to nascent divisomes prior to the other chlamydial divisome proteins assayed, including the PBP2 and PBP3 transpeptidases, and MreB and MreC. Knocking down FtsK prevents divisome assembly in Ct and inhibits cell division and septal peptidoglycan synthesis. We further show that MreB does not function like FtsZ and serve as a scaffold for the assembly of the Ct divisome. Rather, MreB is one of the last proteins recruited to the chlamydial divisome, and it is necessary for the formation of septal peptidoglycan rings. Our studies illustrate the critical role of chlamydial FtsK in coordinating divisome assembly and peptidoglycan synthesis in this obligate intracellular bacterial pathogen.

microbiology↗

bioRxiv2022

Localized Cardiolipin Synthesis is Required for the Assembly of MreB During the Polarized Cell Division of Chlamydia trachomatis

Pathogenic Chlamydia species are coccoid bacteria that use the rod-shape determining protein MreB to direct septal peptidoglycan synthesis during their polarized cell division process. How the site of polarized budding is determined in this bacterium, where contextual features like membrane curvature are seemingly identical, is unclear. We hypothesized that the accumulation of the phospholipid, cardiolipin (CL), in specific regions of the cell membrane induces localized membrane changes that trigger the recruitment of MreB to the site where the bud will arise. To test this, we ectopically expressed cardiolipin synthase (Cls) and observed a polar distribution for this enzyme in Chlamydia trachomatis. In early division intermediates, Cls was restricted to the bud site where MreB is localized and peptidoglycan synthesis is initiated. The localization profile of Cls throughout division mimicked the distribution of lipids that stain with NAO, a dye that labels CL. Treatment of Chlamydia with 3,6-dinonylneamine (diNN), an antibiotic targeting CL-containing membrane domains, resulted in redistribution of Cls and NAO-staining phospholipids. In addition, MreB localization was altered by diNN treatment, suggesting an upstream regulatory role for CL-containing membranes in directing the assembly of MreB. This hypothesis is consistent with the observation that the clustered localization of Cls is not dependent upon MreB function or peptidoglycan synthesis. Furthermore, expression of a CL-binding protein at the inner membrane of C. trachomatis dramatically inhibited bacterial growth supporting the importance of CL in the division process. Our findings implicate a critical role for localized CL synthesis in driving MreB assembly at the bud site during the polarized cell division of Chlamydia.

microbiology↗

bioRxiv2020

Chlamydia trachomatis Encodes a Dynamic, Ring-Forming Bactofilin Critical for Maintaining Cell Size and Shape

Bactofilins are polymer-forming cytoskeletal proteins that are widely conserved in bacteria. Members of this protein family have diverse functional roles such as orienting subcellular molecular processes, establishing cell polarity, and aiding in cell shape maintenance. Chlamydia species are obligate intracellular bacteria that undergo a developmental cycle alternating between an infectious, non-dividing EB and a non-infectious, dividing RB. As Chlamydia divides by a polarized division process, we hypothesized that BacACT may function to establish polarity in these unique bacteria. Using sequence alignment to the conserved bactofilin domain, we identified a bactofilin ortholog, BacACT, in the obligate intracellular pathogen Chlamydia trachomatis. Utilizing a combination of fusion constructs and high-resolution fluorescence microscopy, we determined that BacACT forms a dynamic, membrane-associated, ring-like structure in Chlamydias replicative RB form. Contrary to our hypothesis, this filamentous ring structure is distinct from the microbes cell division machinery and does not colocalize with septal peptidoglycan or MreB, the major organizer of the bacteriums division complex. Bacterial two-hybrid assays demonstrated BacACT interacts homotypically but does not directly interact with proteins involved in cell division or peptidoglycan biosynthesis. To investigate the function of BacACT in chlamydial development, we constructed a conditional knockdown strain using a newly developed CRISPR interference system. We observed that reducing bacACT expression significantly impacted chlamydial cell size and morphology. Normal RB morphology was restored when an additional copy of BacACT was expressed in trans during knockdown. These data reveal a novel function for chlamydial bactofilin in maintaining cell shape in this obligate intracellular bacterium. IMPORTANCEChlamydia is an ancient, obligate intracellular bacterium with a unique biphasic developmental cycle. As a result of its evolution within the osmotically stable environment of a host cell, Chlamydia has lost its dependence on side-wall peptidoglycan, and maintains only a fraction of the components thought to be required for regulating bacterial cell size and division. As such, very little is known about how Chlamydia species carry out these critical processes in the absence of a stabilizing peptidoglycan layer. In the current study, we identify a novel cytoskeletal element, termed a bactofilin, that is critical for maintaining the morphology of the bacteria. Using state-of-the-art genetic techniques for this organism, we demonstrate that chlamydial bactofilin forms a dynamic ring structure independent of the microbes division machinery and that abrogating its expression level using CRISPR interference results in abnormal morphologic forms. These findings enhance our understanding of chlamydial biology and bactofilins more generally.

microbiology↗

bioRxiv2019

Critical Role for the Unique N-Terminus of Chlamydial MreB in Directing Its Membrane Association and Interaction with Elements of the Divisome

Chlamydiae lack the conserved central coordinator protein of cell division FtsZ, a tubulin-like homolog. Current evidence indicates Chlamydia uses the actin-like homolog, MreB, to substitute for the role of FtsZ. Interestingly, we observed MreB as a ring at the septum in dividing cells of Chlamydia. We hypothesize that MreB, to substitute for FtsZ in Chlamydia, must possess unique properties compared to canonical MreB orthologs. Sequence differences between chlamydial MreB and orthologs in other bacteria revealed that chlamydial MreB possesses an extended N-terminal region and the conserved amphipathic helix found in other bacterial MreBs. The extended N-terminal region was sufficient to restore the localization of a truncated E. coli MreB mutant lacking its amphipathic helix to the membrane and was crucial for interactions with cell division components RodZ and FtsK, though the region was not required for homotypic interactions. Importantly, the N-terminal region was sufficient to direct GFP to the membrane when expressed in Chlamydia. A mutant N-terminal region with reduced amphipathicity was unable to perform these functions. From these data, the extended N-terminal region of chlamydial MreB is critical for localization and interactions of this protein. Our data provide mechanistic support for chlamydial MreB to serve as a substitute for FtsZ. ImportanceChlamydia trachomatis is an obligate intracellular pathogen, causing sexual transmitted diseases and trachoma. Studying chlamydial physiology, especially its cell division mechanism, is important for developing novel therapeutic strategies for the treatment of these diseases. Since chlamydial cell division has unique features, including a polarized cell division process independent of FtsZ, a canonical cell division coordinator, studying the subject is helpful for understanding undefined aspects of chlamydial growth. In this study, we characterized MreB, a substitute for FtsZ, as a cell division coordinator. It forms a filamentous ring at the septum, like FtsZ in E. coli. We show that the localization of MreB is dependent upon the amphipathic nature of its extended N-terminus. Furthermore, this region is crucial for its interaction with other proteins involved in cell division. Given these results, chlamydial MreB may function as a scaffold for cell divisome proteins at the septum and regulate cell division in this organism.

microbiology↗