Chlamydia Trachomatis Inclusion Research Articles

Methylene Blue-Loaded NanoMOFs: Accumulation in Chlamydia trachomatis Inclusions and Light/Dark Antibacterial Effects.

Metal-organic framework nanoparticles (nanoMOFs) are promising nanomaterials for biomedical applications. Some of them, including biodegradable porous iron carboxylates are proposed for encapsulation and delivery of antibiotics. Due to the high drug loading capacity and fast internalization kinetics, nanoMOFs are more beneficial for the treatment of intracellular bacterial infections compared to free antibacterial drugs, which poorly accumulate inside the cells because of the inability to cross membrane barriers or have low intracellular retention. However, nanoparticle internalization does not ensure their accumulation in the cell compartment that shelters a pathogen. This study shows the availability of MIL-100(Fe)-based MOF nanoparticles to co-localize with Chlamydia trachomatis, an obligate intracellular bacterium, in the infected RAW264.7 macrophages. Furthermore, nanoMOFs loaded with photosensitizer methylene blue (MB) exhibit complete photodynamic inactivation of C. trachomatis growth. Simultaneous infection and treatment of RAW264.7 cells with empty nanoMOFs resulted in a bacterial load reduction from 100 to 36% that indicates an intrinsic anti-chlamydial effect of this iron-containing nanomaterial. Thus, our findings suggest the use of iron-based nanoMOFs as a promising drug delivery platform, which contributes to antibacterial effect, for the treatment of chlamydial infections.

Homologues of the Chlamydia trachomatis and Chlamydia muridarum Inclusion Membrane Protein IncS Are Interchangeable for Early Development but Not for Inclusion Stability in the Late Developmental Cycle.

Chlamydia trachomatis is an obligate intracellular bacterium, which undergoes a biphasic developmental cycle inside a vacuole termed the inclusion. Chlamydia-specific effector proteins embedded into the inclusion membrane, the Inc proteins, facilitate inclusion interaction with cellular organelles. A subset of Inc proteins engages with specific host factors at the endoplasmic reticulum (ER)-inclusion membrane contact site (MCS), which is a discrete point of contact between the inclusion membrane and the endoplasmic reticulum (ER). Here, we report that the C. trachomatis Inc protein CTL0402/IncSCt is a novel component of the ER-inclusion MCS that specifically interacts with and recruits STIM1, a previously identified host component of the ER-inclusion MCS with an unassigned interacting partner at the inclusion membrane. In comparison, the Chlamydia muridarum IncS homologue (TC0424/IncSCm) does not interact with or recruit STIM1 to the inclusion, indicating species specificity. To further investigate IncS function and overcome the recently reported early developmental defect of the incS mutant, we achieved temporal complementation by expressing IncS exclusively during the early stages of the developmental cycle. Additionally, we used allelic exchange to replace the incSCt open reading frame with incSCm in the C. trachomatis chromosome. Inclusions harboring either of these strains progressed through the developmental cycle but were STIM1 negative and displayed increased inclusion lysis 48 h postinfection. Expression of incSCt in trans complemented these phenotypes. Altogether, our results indicate that IncS is necessary and sufficient to recruit STIM1 to C. trachomatis inclusion and that IncS plays an early developmental role conserved in C. trachomatis and C. muridarum and a late role in inclusion stability specific to C. trachomatis. IMPORTANCE Obligate intracellular pathogens strictly rely on the host for replication. Specialized pathogen-encoded effector proteins play a central role in sophisticated mechanisms of host cell manipulation. In Chlamydia, a subset of these effector proteins, the inclusion membrane proteins, are embedded in the membrane of the vacuole in which the bacteria replicate. Chlamydia encodes 50 to 100 putative Inc proteins. Many are conserved among species, including the human and mouse pathogens Chlamydia trachomatis and Chlamydia muridarum, respectively. However, whether the function(s) of Inc proteins is indeed conserved among species is poorly understood. Here, we characterized the function of the Inc protein IncS conserved in C. trachomatis and C. muridarum. Our work reveals that a single effector protein can play multiple functions at various stages of the developmental cycle. However, these functions are not necessarily conserved across species, suggesting a complex evolutionary path among Chlamydia species.

The bacterial effector GarD shields Chlamydia trachomatis inclusions from RNF213-mediated ubiquitylation and destruction

Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infections and a major threat to women's reproductive health in particular. This obligate intracellular pathogen resides and replicates within a cellular compartment termed an inclusion, where it is sheltered by unknown mechanisms from gamma-interferon (IFNγ)-induced cell-autonomous host immunity. Through a genetic screen, we uncovered the Chlamydia inclusion membrane protein gamma resistance determinant (GarD) as a bacterial factor protecting inclusions from cell-autonomous immunity. In IFNγ-primed human cells, inclusions formed by garD loss-of-function mutants become decorated with linear ubiquitin and are eliminated. Leveraging cellular genome-wide association data, we identified the ubiquitin E3 ligase RNF213 as a candidate anti-Chlamydia protein. We demonstrate that IFNγ-inducible RNF213 facilitates the ubiquitylation and destruction of GarD-deficient inclusions. Furthermore, we show that GarD operates as a cis-acting stealth factor barring RNF213 from targeting inclusions, thus functionally defining GarD as an RNF213 antagonist essential for chlamydial growth during IFNγ-stimulated immunity.

The Chlamydia trachomatis Inclusion Membrane Protein CTL0390 Mediates Host Cell Exit via Lysis through STING Activation.

The obligate intracellular bacterium Chlamydia trachomatis is the causative agent of the most frequently reported bacterial sexually transmitted disease. Upon internalization into host cells, C. trachomatis remains within a membrane-bound compartment known as an inclusion, where it undergoes its developmental cycle. After completion of this cycle, bacteria exit the host cell. One mechanism of exit is lysis, whereby the inclusion and host cell rupture to release bacteria; however, the mechanism of lysis is not well characterized. A subset of C. trachomatis effectors, known as inclusion membrane proteins (Inc), are embedded within the inclusion membrane to facilitate host cell manipulation. The functions of many Inc proteins are unknown. We sought to characterize the Inc protein CTL0390. We determined that CTL0390 is expressed throughout the developmental cycle and that its C-terminal tail is exposed to the cytosol. To investigate the function of CTL0390, we generated a ctl0390 mutant complemented with ctl0390 on a plasmid. Loss of CTL0390 did not affect infectious progeny production but resulted in a reduction in lysis. Overexpression of CTL0390 induced premature lysis and host nuclear condensation, the latter of which could be reduced upon inhibition of the cGAS-STING DNA sensing pathway. Infection with the clt0390 mutant led to reduced Golgi translocation of STING, and chemical and genetic approaches to inactivate STING revealed that STING plays a role in lysis in a CTL0390-dependent manner. Together, these results reveal a role for CTL0390 in bacterial exit via lysis at late stages of the Chlamydia developmental cycle and through STING activation.

The Chlamydia trachomatis inclusion membrane protein CT006 associates with lipid droplets in eukaryotic cells.

Chlamydia trachomatis causes genital and ocular infections in humans. This bacterial pathogen multiplies exclusively within host cells in a characteristic vacuole (inclusion) and delivers proteins such as inclusion membrane proteins (Incs) into the host cell. Here, we identified CT006 as a novel C. trachomatis protein that when expressed ectopically eukaryotic cells can associate with lipid droplets (LDs). A screen using Saccharomyces cerevisiae identified two Incs causing vacuolar protein sorting defects and seven Incs showing tropism for eukaryotic organelles. Ectopic expression in yeast and mammalian cells of genes encoding different fragments of CT006 revealed tropism for the endoplasmic reticulum and LDs. We identified a LD-targeting region within the first 88 amino acid residues of CT006, and positively charged residues important for this targeting. Comparing with the parental wild-type strain, cells infected by a newly generated C. trachomatis strain overproducing CT006 with a double hemagglutinin tag showed a slight increase in the area occupied by LDs within the inclusion region. However, we could not correlate this effect with the LD-targeting regions within CT006. We further showed that both the amino and carboxy-terminal regions of CT006, flanking the Inc-characteristic bilobed hydrophobic domain, are exposed to the host cell cytosol during C. trachomatis infection, supporting their availability to interact with host cell targets. Altogether, our data suggest that CT006 might participate in the interaction of LDs with C. trachomatis inclusions.

HGBP1 Coordinates Chlamydia Restriction and Inflammasome Activation through Sequential GTP Hydrolysis

Human guanylate binding protein 1 (hGBP1) belongs to the dynamin superfamily of GTPases and conveys host defense against intracellular bacteria and parasites. During infection, hGBP1 is recruited to pathogen-containing vacuoles, such as Chlamydia trachomatis inclusions, restricts pathogenic growth, and induces the activation of the inflammasome pathway. hGBP1 has a unique catalytic activity to hydrolyze guanosine triphosphate (GTP) to guanosine monophosphate (GMP) in two consecutive cleavage steps. However, the functional significance of this activity in host defense remains elusive. Here, we generate a structure-guided mutant that specifically abrogates GMP production, while maintaining fast cooperative GTP hydrolysis. Complementation experiments in human monocytes/macrophages show that hGBP1-mediated GMP production is dispensable for restricting Chlamydia trachomatis growth but is necessary for inflammasome activation. Mechanistically, GMP is catabolized to uric acid, which in turn activates the NLRP3 inflammasome. Our study demonstrates that the unique enzymology of hGBP1 coordinates bacterial growth restriction and inflammasome signaling.

A meta-analysis of affinity purification-mass spectrometry experimental systems used to identify eukaryotic and chlamydial proteins at the Chlamydia trachomatis inclusion membrane

The obligate intracellular bacterial pathogen, Chlamydia trachomatis, develops within a membrane-bound vacuole termed the inclusion. Affinity purification-mass spectrometry (AP-MS) experiments to study the interactions that occur at the chlamydial inclusion membrane have been performed and, more recently, combined with advances in C. trachomatis genetics. However, each of the four AP-MS published reports used either different experimental approaches or statistical tools to identify proteins that localize at the inclusion. We critically analyzed each experimental approach and performed a meta-analysis of the reported statistically significant proteins for each study, finding that only a few eukaryotic proteins were commonly identified between all four experimental approaches. The two similarly conducted in vivo labeling studies were compared using the same statistical analysis tool, Significance Analysis of INTeractome (SAINT), which revealed a disparity in the number of significant proteins identified by the original analysis. We further examined methods to identify potential background contaminant proteins that remain after statistical analysis. Overall, this meta-analysis highlights the importance of carefully controlling and analyzing the AP-MS data so that pertinent information can be obtained from these various AP-MS experimental approaches. This study provides important guidelines and considerations for using this methodology to study intracellular pathogens residing within a membrane-bound compartment. SignificanceChlamydia trachomatis, an obligate intracellular pathogen, grows within a membrane-bound vacuole termed the inclusion. The inclusion is studded with bacterial membrane proteins that likely orchestrate numerous interactions with the host cell. Although maintenance of the intracellular niche is vital, an understanding of the host-pathogen interactions that occur at the inclusion membrane is limited by the difficulty in purifying membrane protein fractions from infected host cells. The experimental procedures necessary to solubilize hydrophobic proteins fail to maintain transient protein-protein interactions. Advances in C. trachomatis genetics has allowed us and others to use various experimental approaches in combination with affinity purification mass spectrometry (AP-MS) to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. For the first time, two groups have published AP-MS studies using the same tool, the ascorbate peroxidase proximity labeling system (APEX2), which overcomes past experimental limitations because membrane protein interactions are labeled in vivo in the context of infection. The utility of this system is highlighted by its ability to study chlamydial type III secreted inclusion membrane protein (Inc) interactions. Incs act as the mediators of host-pathogen interactions at the inclusion during C. trachomatis infection. When carefully controlled and analyzed, the data obtained can yield copious amounts of useful information. Here, we critically analyzed four previously published studies, including statistical analysis of AP-MS datasets related to Chlamydia-host interactions, to contextualize the data and to identify the best practices in interpreting these types of complex outputs.

Characterization of the function of murine guanylate-binding protein 9 in the context of bacterial and parasitic infections

Abstract Interferon-γ (IFNγ) signaling induces high expression of immunomodulatory molecules in most cell types. Among these molecules, the members of the murine guanylate-binding protein (mGBP) family comprise a group of eleven proteins (mGBP1-mGBP11) shown to have important functions in cell-autonomous immunity. Upon IFNγ stimulation, mGBPs localize in vesicle-like structures distributed within the cytoplasm. Previous studies have shown that after infection with several intracellularly replicating pathogens, mGBPs rapidly relocate towards the pathogen-containing vacuoles (PCV), such as the parasitophorous vacuole (PV) of Toxoplasma gondii or the inclusions of Chlamydia trachomatis. Accumulation of mGBP2 at the PV membrane leads to loss of membrane integrity and subsequent restriction of parasite replication. Although mGBP proteins share a high sequence identity, differences in localization frequencies could be detected. For instance, mGBP1 and mGBP2 show the highest frequencies of recruitment to both Toxoplasma and Chlamydia PCVs. However, mGBP9 recruitment was more frequent to chlamydial inclusions than to T. gondii PVs. Thus, the importance of individual mGBPs might vary depending on the type of the pathogen. To analyze these findings in more detail, we started with the generation of mgbp9−/− cell- and mouse lines. Therefore, the CRISPR/Cas9 gene editing system with homologous directed integration of a single-stranded oligodesoxynucleotide is used to generate mGBP9 deficient ES cells for mouse and cell line development. In-depth analyzes of mgbp9−/− cells and mice in T. gondii and C. trachomatis infection will lead to a better understanding of immunity against intracellular pathogens.

Proximity-dependent proteomics of the Chlamydia trachomatis inclusion membrane reveals functional interactions with endoplasmic reticulum exit sites.

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection, responsible for millions of infections each year. Despite this high prevalence, the elucidation of the molecular mechanisms of Chlamydia pathogenesis has been difficult due to limitations in genetic tools and its intracellular developmental cycle. Within a host epithelial cell, chlamydiae replicate within a vacuole called the inclusion. Many Chlamydia–host interactions are thought to be mediated by the Inc family of type III secreted proteins that are anchored in the inclusion membrane, but their array of host targets are largely unknown. To investigate how the inclusion membrane proteome changes over the course of an infected cell, we have adapted the APEX2 system of proximity-dependent biotinylation. APEX2 is capable of specifically labeling proteins within a 20 nm radius in living cells. We transformed C. trachomatis to express the enzyme APEX2 fused to known inclusion membrane proteins, allowing biotinylation and purification of inclusion-associated proteins. Using quantitative mass spectrometry against APEX2 labeled samples, we identified over 400 proteins associated with the inclusion membrane at early, middle, and late stages of epithelial cell infection. This system was sensitive enough to detect inclusion interacting proteins early in the developmental cycle, at 8 hours post infection, a previously intractable time point. Mass spectrometry analysis revealed a novel, early association between C. trachomatis inclusions and endoplasmic reticulum exit sites (ERES), functional regions of the ER where COPII-coated vesicles originate. Pharmacological and genetic disruption of ERES function severely restricted early chlamydial growth and the development of infectious progeny. APEX2 is therefore a powerful in situ approach for identifying critical protein interactions on the membranes of pathogen-containing vacuoles. Furthermore, the data derived from proteomic mapping of Chlamydia inclusions has illuminated an important functional role for ERES in promoting chlamydial developmental growth.

Genetic Inactivation of Chlamydia trachomatis Inclusion Membrane Protein CT228 Alters MYPT1 Recruitment, Extrusion Production, and Longevity of Infection.

Chlamydia trachomatis is an obligate intracellular pathogen with global health and economic impact. Upon infection, C. trachomatis resides within a protective niche, the inclusion, wherein it replicates and usurps host cell machinery and resources. The inclusion membrane is the key host-pathogen interface that governs specific protein-protein interactions to manipulate host signaling pathways. At the conclusion of the infection cycle, C. trachomatis exits the host cell via lysis or extrusion. Extrusion depends on the phosphorylation state of myosin light chain 2 (MLC2); the extent of phosphorylation is determined by the ongoing opposing activities of myosin phosphatase (MYPT1) and myosin kinase (MLCK). Previously, it was shown that MYPT1 is recruited to the inclusion and interacts with CT228 for regulation of host cell egress. In this study, we generated a targeted chromosomal mutation of CT228 (L2-ΔCT228) using the TargeTron system and demonstrate a loss of MYPT1 recruitment and increase in extrusion production in vitro. Mutation of CT228 did not affect chlamydial growth in cell culture or recruitment of MLC2. Moreover, we document a delay in clearance of L2-ΔCT228 during murine intravaginal infection as well as a reduction in systemic humoral response, relative to L2-wild type. Taken together, the data suggest that loss of MYPT1 recruitment (as a result of CT228 disruption) regulates the degree of host cell exit via extrusion and affects the longevity of infection in vivo.

The Chlamydia trachomatis Plasmid and CT135 Virulence Factors Are Not Essential for Genital Tract Infection or Pathology in Female Pig-Tailed Macaques.

ABSTRACTThe Chlamydia trachomatis plasmid and inclusion membrane protein CT135 are virulence factors in the pathogenesis of murine female genital tract infection. To determine if these virulence factors play a similar role in female nonhuman primates, we infected pig-tailed macaques with the same C. trachomatis strains shown to be important in the murine model. Wild-type C. trachomatis and its isogenic mutant strain deficient in both plasmid and CT135 were used to infect macaques. Macaques were given primary and repeated cervicovaginal challenges with the wild-type and mutant strains. The infection rate, infection duration, and antibody response were similar among macaques infected with both strains. Unexpectedly, colposcopy, laparoscopy, and histologic analysis revealed no substantial genital tract pathology following either primary or repeated cervicovaginal challenges. Cytokine analysis of cervicovaginal secretions from both challenged groups revealed low concentrations of interleukin 1β (IL-1β) and elevated levels of the interleukin 1 receptor agonist (IL-1RA). We propose that an imbalance of IL-1β and IL-1RA in macaques is the reason for the mild inflammatory responses observed in infected urogenital tissues. Thus, understanding the pathobiology of chlamydial infection requires a better understanding of host epigenetic and chlamydial genetic factors. Our findings also have implications for understanding the high frequency of asymptomatic infections in humans.

Chlamydia trachomatis inclusion membrane protein MrcA interacts with the inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) to regulate extrusion formation.

Chlamydia trachomatis is an obligate intracellular bacterium that replicates within a vacuole termed an inclusion. At the end of their intracellular developmental cycle, chlamydiae are released either by lysis of the host cell or extrusion of the intact inclusion. The inclusion membrane is extensively modified by the insertion of type III secreted inclusion membrane proteins, Incs, which contribute to inclusion membrane structure and facilitate host-pathogen interactions. An interaction was identified between the inclusion membrane protein, MrcA, and the Ca2+ channel inositol-1,4,5-trisphosphate receptor, type 3 (ITPR3). ITPR3 was recruited and localized to active Src-family-kinase rich microdomains on the inclusion membrane as was the Ca2+ sensor, STIM1. Disruption of MrcA by directed mutagenesis resulted in loss of ITPR3 recruitment and simultaneous reduction of chlamydial release by extrusion. Complementation of MrcA restored ITPR3 recruitment and extrusion. Inhibition of extrusion was also observed following siRNA depletion of host ITPR3 or STIM1. Chlamydial extrusion was also inhibited by the calcium chelator BAPTA-AM. Each of these treatments resulted in a concomitant reduction in phosphorylation of the myosin regulatory light chain (MLC2) and a loss of myosin motor activity at the end of the developmental cycle which is consistent with the reduced extrusion formation. These studies suggest that Ca2+ signaling pathways play an important role in regulation of release mechanisms by C. trachomatis.

Broad recruitment of mGBP family members to Chlamydia trachomatis inclusions.

Chlamydia, the most common sexually transmitted pathogen, is an exquisitely adapted Gram-negative obligate intracellular bacterium. Intracellular Chlamydia trachomatis replicate in a specialized vacuole, termed inclusion, which shields the bacterium from antimicrobial immunity of the host cells and acts as a signalling interface. Previously it was shown that members of the interferon induced guanylate binding protein (mGBP) family, in particular murine GBP1 and mGBP2, were found to accumulate at the bacterial inclusions, similar to previously published recruitment of GBPs to the parasitophorous vacuole of Toxoplasma gondii. Here, we provide a wide comparison of mGBPs roles within the host cell in the context of Chlamydia and Toxoplasma infection. By confocal microscopy on fixed and living infected cells we show localization of mGBP3, mGBP6, mGBP7, mGBP9, and mGBP10, in addition to mGBP1 and mGBP2, at chlamydia inclusions. In time lapse videos using GFP expressing Chlamydia we show rapid and transient dynamics of mGBP9 accumulation onto chlamydia inclusions. Taken together this study reveals a broad activation of mGBP recruitment towards Chlamydia trachomatis inclusions after infection and provides evidence for time limited action of mGBP9 at the chlamydia inclusion.

Ageloline A, new antioxidant and antichlamydial quinolone from the marine sponge-derived bacterium Streptomyces sp. SBT345

A new chlorinated quinolone, ageloline A, was isolated from the broth culture of Streptomyces sp. SBT345 that was cultivated from the Mediterranean sponge Agelas oroides. The structure of this compound was determined by spectroscopic analysis including 1D and 2D NMR as well as HR-ESI-MS experiments. Ageloline A exhibited antioxidant potential using cell-free and cell-based assays and was further able to reduce oxidative stress and genomic damage induced by the oxidative stress inducer 4-nitroquinoline-1-oxide (NQO). Moreover, ageloline A inhibited the formation and growth of Chlamydia trachomatis inclusion in a dose-dependent manner with an IC50 value of 9.54±0.36μM. Interestingly, experimental data showed that the antichlamydial activity of ageloline A might be related to its antioxidant potential. These results demonstrate that sponge-associated actinomycetes are rich sources for natural products with new pharmacological activities and relevance to drug discovery.

Differential Translocation of Host Cellular Materials into the Chlamydia trachomatis Inclusion Lumen during Chemical Fixation.

Chlamydia trachomatis manipulates host cellular pathways to ensure its proliferation and survival. Translocation of host materials into the pathogenic vacuole (termed ‘inclusion’) may facilitate nutrient acquisition and various organelles have been observed within the inclusion, including lipid droplets, peroxisomes, multivesicular body components, and membranes of the endoplasmic reticulum (ER). However, few of these processes have been documented in living cells. Here, we survey the localization of a broad panel of subcellular elements and find ER, mitochondria, and inclusion membranes within the inclusion lumen of fixed cells. However, we see little evidence of intraluminal localization of these organelles in live inclusions. Using time-lapse video microscopy we document ER marker translocation into the inclusion lumen during chemical fixation. These intra-inclusion ER elements resist a variety of post-fixation manipulations and are detectable via immunofluorescence microscopy. We speculate that the localization of a subset of organelles may be exaggerated during fixation. Finally, we find similar structures within the pathogenic vacuole of Coxiella burnetti infected cells, suggesting that fixation-induced translocation of cellular materials may occur into the vacuole of a range of intracellular pathogens.

Chlamydia trachomatis inclusion membrane protein CT850 interacts with the dynein light chain DYNLT1 (Tctex1)

Chlamydia trachomatis actively subverts the minus-end directed microtubule motor, dynein, to traffic along microtubule tracks to the Microtubule Organizing Center (MTOC) where it remains within a membrane bound replicative vacuole for the duration of its intracellular development. Unlike most substrates of the dynein motor, disruption of the dynactin cargo-linking complex by over-expression of the p50 dynamitin subunit does not inhibit C. trachomatis transport. A requirement for chlamydial protein synthesis to initiate this process suggests that a chlamydial product supersedes a requirement for p50 dynamitin. A yeast 2-hybrid system was used to screen the chlamydia inclusion membrane protein CT850 against a HeLa cell cDNA library and identified an interaction with the dynein light chain DYNLT1 (Tctex1). This interaction was at least partially dependent upon an (R/K-R/K-X-X-R/K) motif that is characteristic of DYNLT1 binding domains. CT850 expressed ectopically in HeLa cells localized at the MTOC and this localization is similarly dependent upon the predicted DYNLT1 binding domain. Furthermore, DYNLT1 is enriched at focal concentrations of CT850 on the chlamydial inclusion membrane that are known to interact with dynein and microtubules. Depletion of DYNLT1 disrupts the characteristic association of the inclusion membrane with centrosomes. Collectively, the results suggest that CT850 interacts with DYNLT1 to promote appropriate positioning of the inclusion at the MTOC.

Expansion of the Chlamydia trachomatis inclusion does not require bacterial replication

Chlamydia trachomatis replication takes place inside of a host cell, exclusively within a vacuole known as the inclusion. During an infection, the inclusion expands to accommodate the increasing numbers of C. trachomatis. However, whether inclusion expansion requires bacterial replication and/or de novo protein synthesis has not been previously investigated in detail. Therefore, using a chemical biology approach, we herein investigated C. trachomatis inclusion expansion under varying conditions in vitro. Under normal cell culture conditions, inclusion expansion correlated with C. trachomatis replication. When bacterial replication was inhibited using KSK120, an inhibitor that targets C. trachomatis glucose metabolism, inclusions expanded even in the absence of bacterial replication. In contrast, when bacterial protein synthesis was inhibited using chloramphenicol, expansion of inclusions was blocked. Together, these data suggest that de novo protein synthesis is necessary, whereas bacterial replication is dispensable for C. trachomatis inclusion expansion.

Septins arrange F-actin-containing fibers on the Chlamydia trachomatis inclusion and are required for normal release of the inclusion by extrusion.

ABSTRACTChlamydia trachomatis is an obligate intracellular human pathogen that grows inside a membranous, cytosolic vacuole termed an inclusion. Septins are a group of 13 GTP-binding proteins that assemble into oligomeric complexes and that can form higher-order filaments. We report here that the septins SEPT2, -9, -11, and probably -7 form fibrillar structures around the chlamydial inclusion. Colocalization studies suggest that these septins combine with F actin into fibers that encase the inclusion. Targeting the expression of individual septins by RNA interference (RNAi) prevented the formation of septin fibers as well as the recruitment of actin to the inclusion. At the end of the developmental cycle of C. trachomatis, newly formed, infectious elementary bodies are released, and this release occurs at least in part through the organized extrusion of intact inclusions. RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells. The data suggest that a higher-order structure of four septins is involved in the recruitment or stabilization of the actin coat around the chlamydial inclusion and that this actin recruitment by septins is instrumental for the coordinated egress of C. trachomatis from human cells. The organization of F actin around parasite-containing vacuoles may be a broader response mechanism of mammalian cells to the infection by intracellular, vacuole-dwelling pathogens.

Chlamydia trachomatis Inclusion Membrane Protein CT228 Recruits Elements of the Myosin Phosphatase Pathway to Regulate Release Mechanisms

Chlamydia trachomatis replicates within a membrane-bound compartment termed an inclusion. The inclusion membrane is modified by the insertion of multiple proteins known as Incs. In a yeast two-hybrid screen, an interaction was found between the inclusion membrane protein CT228 and MYPT1, a subunit of myosin phosphatase. MYPT1 was recruited peripherally around the inclusion, whereas the phosphorylated, inactive form was localized to active Src-family kinase-rich microdomains. Phosphorylated myosin light chain 2 (MLC2), myosin light chain kinase (MLCK), myosin IIA, and myosin IIB also colocalized with inactive MYPT1. The role of these proteins was examined in the context of host-cell exit mechanisms (i.e., cell lysis and extrusion of intact inclusions). Inhibition of myosin II or small interfering RNA depletion of myosin IIA, myosin IIB, MLC2, or MLCK reduced chlamydial extrusion, thus favoring lytic events as the primary means of release. These studies provide insights into the regulation of egress mechanisms by C.trachomatis.

Chlamydia trachomatis hijacks intra-Golgi COG complex-dependent vesicle trafficking pathway

Chlamydia spp. are obligate intracellular bacteria that replicate inside the host cell in a bacterial modified unique compartment called the inclusion. As other intracellular pathogens, chlamydiae exploit host membrane trafficking pathways to prevent lysosomal fusion and to acquire energy and nutrients essential for their survival and replication. The Conserved Oligomeric Golgi (COG) complex is a ubiquitously expressed membrane-associated protein complex that functions in a retrograde intra-Golgi trafficking through associations with coiled-coil tethers, SNAREs, Rabs and COPI proteins. Several COG complex-interacting proteins, including Rab1, Rab6, Rab14 and Syntaxin 6 are implicated in chlamydial development. In this study, we analysed the recruitment of the COG complex and GS15-positive COG complex-dependent vesicles to Chlamydia trachomatis inclusion and their participation in chlamydial growth. Immunofluorescent analysis revealed that both GFP-tagged and endogenous COG complex subunits associated with inclusions in a serovar-independent manner by 8 h post infection and were maintained throughout the entire developmental cycle. Golgi v-SNARE GS15 was associated with inclusions 24 h post infection, but was absent on the mid-cycle (8 h) inclusions, indicating that this Golgi SNARE is directed to inclusions after COG complex recruitment. Silencing of COG8 and GS15 by siRNA significantly decreased infectious yield of chlamydiae. Further, membranous structures likely derived from lysed bacteria were observed inside inclusions by electron microscopy in cells depleted of COG8 or GS15. Our results showed that C. trachomatis hijacks the COG complex to redirect the population of Golgi-derived retrograde vesicles to inclusions. These vesicles likely deliver nutrients that are required for bacterial development and replication.