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Epstein-Barr virus (EBV) is a major cause of infectious mononucleosis (IM), characterized by fever, fatigue, sore throat, lymphadenopathy, atypical lymphocytosis, and elevated liver enzymes. However, ascites is a rare complication associated with IM. We present a rare case of IM with ascites and peritonitis in a patient who underwent a peritoneal biopsy. A 20-year-old woman presented with fatigue and abdominal distension. Laboratory examination revealed atypical lymphocytes in peripheral blood (54%) and elevated liver enzymes. EBV serological tests revealed a recent primary infection (EBV VCA IgM 1:160). Computed tomography revealed moderate ascites and peritonitis. Adenocarcinoma was suspected based on the ascites' cytology. Considering possible complications of IM and adenocarcinoma, a laparoscopic biopsy was performed. Histological findings of biopsy specimens from the peritoneum, omentum, and fimbria of the fallopian tube demonstrated severe inflammatory cell infiltration and focal aggregation of large EBV-encoded RNA-1 (EBER1)-positive B cells, mimicking EBV-positive polymorphous B-cell lymphoproliferative disorder. Furthermore, intracytoplasmic inclusion bodies of Chlamydia trachomatis were observed by immunohistochemistry. Real-time polymerase chain reaction detected C. trachomatis in cervical secretions. Two months after laparoscopy, ascites decreased, and the diagnosis was IM-associated peritonitis with C. trachomatis infection. IM should be considered as a differential diagnosis in young patients with ascites., (© 2024 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Chlamydia trachomatis is a clinically important bacterium that infects epithelial cells of the genitourinary and respiratory tracts and the eye. These differentiated cells are in a quiescent growth state and have a surface organelle called a primary cilium, but the standard Chlamydia cell culture infection model uses cycling cells that lack primary cilia. To investigate if these differences are relevant, we performed infections with host cells that have a primary cilium. We found that C. trachomatis caused progressive loss of the primary cilium that was prevented by disrupting Aurora A (AurA), HDAC6 or calmodulin, which are components of the cellular cilia disassembly pathway. Stabilization of the primary cilium by targeting this pathway caused a large reduction in infectious progeny although there were no changes in chlamydial inclusion growth, chlamydial replication or the ultrastructural appearance of dividing and infectious forms (RBs and EBs, respectively). Thus, the presence of a primary cilium interfered with the production of infectious EBs at a late step in the developmental cycle. C. trachomatis infection also induced quiescent cells to re-enter the cell cycle, as detected by EdU incorporation in S-phase, and Chlamydia-induced cilia disassembly was necessary for cell cycle re-entry. This study therefore describes a novel host-pathogen interaction in which the primary cilium limits a productive Chlamydia infection, and the bacterium counteracts this host cell defense by activating the cellular cilia disassembly pathway., Competing Interests: The authors have declared that no competing interests exists., (Copyright: © 2024 Ekka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chlamydia trachomatis, an obligate intracellular bacterium with limited metabolic capabilities, possesses the futalosine pathway for menaquinone biosynthesis. Futalosine pathway enzymes have promise as narrow-spectrum antibiotic targets, but the activity and essentiality of chlamydial menaquinone biosynthesis have yet to be established. In this work, menaquinone-7 (MK-7) was identified as a C. trachomatis-produced quinone through liquid chromatography-tandem mass spectrometry. An immunofluorescence-based assay revealed that treatment of C. trachomatis-infected HeLa cells with the futalosine pathway inhibitor docosahexaenoic acid (DHA) reduced inclusion number, inclusion size, and infectious progeny. Supplementation with MK-7 nanoparticles rescued the effect of DHA on inclusion number, indicating that the futalosine pathway is a target of DHA in this system. These results open the door for menaquinone biosynthesis inhibitors to be pursued in antichlamydial development., (© 2021 Federation of European Biochemical Societies.)

Epidemiologic, clinical, molecular and translational research findings support an interrelationship between Chlamydia trachomatis, pelvic inflammatory disease (PID), and epithelial ovarian cancer (EOC). Overall, the link between C. trachomatis, PID, and EOC seems to be relatively weak, although nondifferential misclassification bias may have attenuated the results. The predominant tubal origin of EOC and the role of chronic inflammation in tumorigenesis suggest that the association is biologically plausible. Thus, C. trachomatis and PID may represent potential risk factors or risk markers for EOC. However, many steps in this chain of events are still poorly understood and need to be addressed in future studies. Research gaps include time of exposure in relation to the long-term consequences and lag time to EOC. Data of differential risk for EOC between chlamydial and nonchlamydial PID is also needed. Another major research gap has been the absence of high-performance biomarkers for C. trachomatis, PID, and EOC, as well as EOC precursors. Biomarkers for C. trachomatis and PID leading to increased risk of EOC should be developed. If the association is confirmed, C. trachomatis and PID prevention efforts may play a role in reducing the burden of EOC., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: The United States is experiencing a surge in Chlamydia trachomatis (CT) infections representing a critical need to improve sexually transmitted infection (STI) screening and treatment programs. To understand where patients with STIs seek healthcare, we evaluated the relationship between CT infections and the place where individuals report usually receiving healthcare., Methods: Our study used a nationally representative sample from the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2016. The study population is adult patients, aged 18 to 39 years in whom a urine CT screen was obtained. Logistic regression models were used to determine if location of usual healthcare was predictive of a positive urine CT screen result. Models were adjusted for known confounders including age, gender, race/ethnicity, education, and insurance status., Results: In this nationally representative sample (n = 19,275; weighted n = 85.8 million), 1.9% of individuals had a positive urine CT result. Participants reported usually going to the doctor's office (70.3%), "no place" (24.8%), Emergency Department (ED) (3.3%), or "other" place (1.7%) for healthcare. In adjusted models, the predicted probability of having a positive urine CT result is higher (4.9% vs 3.2%, p = 0.022; OR = 1.58) among those that reported the ED as their usual place for healthcare compared to those that reported going to a doctor's office or clinic., Conclusions: Individuals having a positive urine CT screen are associated with using the ED as a usual source for healthcare. Understanding this association has the potential to improve STI clinical and policy interventions as the ED may be a critical site in combatting the record high rates of STIs., Competing Interests: The authors have declared that no competing interests exist.

Chlamydia trachomatis is the most commonly reported sexually transmitted infection in the United States. The high prevalence of infection and lack of a vaccine indicate a critical knowledge gap surrounding the host's response to infection and how to effectively generate protective immunity. The immune response to C. trachomatis is complex, with cells of the adaptive immune system playing a crucial role in bacterial clearance. Here, we discuss the CD4+ and CD8+ T cell response to Chlamydia, the importance of antigen specificity and the role of memory T cells during the recall response. Ultimately, a deeper understanding of protective immune responses is necessary to develop a vaccine that prevents the inflammatory diseases associated with Chlamydia infection., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Chlamydiae are pathogenic intracellular bacteria that cause a wide variety of diseases throughout the globe, affecting the eye, lung, coronary arteries and female genital tract. Rather than by direct cellular toxicity, Chlamydia infection generally causes pathology by inducing fibrosis and scarring that is largely mediated by host inflammation. While a robust immune response is required for clearance of the infection, certain elements of that immune response may also damage infected tissue, leading to, in the case of female genital infection, disease sequelae such as pelvic inflammatory disease, infertility and ectopic pregnancy. It has become increasingly clear that the components of the immune system that destroy bacteria and those that cause pathology only partially overlap. In the ongoing quest for a vaccine that prevents Chlamydia-induced disease, it is important to target mechanisms that can achieve protective immunity while preventing mechanisms that damage tissue. This review focuses on mouse models of genital Chlamydia infection and synthesizes recent studies to generate a comprehensive model for immunity in the murine female genital tract, clarifying the respective contributions of various branches of innate and adaptive immunity to both host protection and pathogenic genital scarring., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

The human pathogen Chlamydia trachomatis targets epithelial cells lining the genital mucosa. We observed that infection of various cell types, including fibroblasts and epithelial cells resulted in the formation of unusually stable and mature focal adhesions that resisted disassembly induced by the myosin II inhibitor, blebbistatin. Superresolution microscopy revealed in infected cells the vertical displacement of paxillin and focal adhesion kinase from the signaling layer of focal adhesions, whereas vinculin remained in its normal position within the force transduction layer. The candidate type III effector TarP, which localized to focal adhesions during infection and when expressed ectopically, was sufficient to mimic both the reorganization and blebbistatin-resistant phenotypes. These effects of TarP, including its localization to focal adhesions, required a post-invasion interaction with the host protein vinculin through a specific domain at the C terminus of TarP. This interaction is repurposed from an actin-recruiting and -remodeling complex to one that mediates nanoarchitectural and dynamic changes of focal adhesions. The consequence of Chlamydia -stabilized focal adhesions was restricted cell motility and enhanced attachment to the extracellular matrix. Thus, via a novel mechanism, Chlamydia inserts TarP within focal adhesions to alter their organization and stability., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Pedrosa et al.)

Chlamydia trachomatis is the leading cause of sexually transmitted infections that may progress to pelvic inflammatory disease and infertility. No effective vaccine exists for Chlamydia , nor are there biomarkers available that readily predict disease progression. In this cross-sectional pilot study, we recruited symptomatic and asymptomatic women with C. trachomatis (CT) infection and asymptomatic, uninfected control women from an urban sexually transmitted disease clinic to determine if there were differences in microRNA (miRNA) expression. Infected women with signs and/or symptoms (CTSS) have distinct miRNA profiles compared to asymptomatic infected women (CTNS). In the CTSS group, miR-142 and -147 showed 2.2- to 6.9-fold increases in expression. In the CTNS group, miR-449c, -6779, -519d, -449a, and -2467 showed 3.9- to 9.0-fold increases in expression. In the CTNS group, cyclins and cell cycle regulation and IL-17 pathways were likely downregulated, while the same signaling pathways were upregulated in the CTSS group. In addition, in the CTSS group, additional inflammatory pathways associated with TNFR1 and IL-8 appear to be upregulated. The miRNA expression patterns differ between CT-infected symptomatic and asymptomatic women, and these differences may warrant further study., (Copyright © 2020 American Society for Microbiology.)

Chlamydia bacteria are obligate intracellular pathogens which can cause a variety of disease in humans and other vertebrate animals. To successfully complete its life cycle, Chlamydia must evade both intracellular innate immune responses and adaptive cytotoxic T cell responses. Here, we report on the role of the chlamydial lipooligosaccharide (LOS) in evading the immune response. Chlamydia infection is known to block the induction of apoptosis. However, when LOS synthesis was inhibited during Chlamydia trachomatis infection, HeLa cells regained susceptibility to apoptosis induction following staurosporine treatment. Additionally, the delivery of purified LOS to the cytosol of cells increased the levels of the antiapoptotic protein survivin. An increase in survivin levels was also detected following C. trachomatis infection, which was reversed by blocking LOS synthesis. Interestingly, while intracellular delivery of lipopolysaccharide (LPS) derived from Escherichia coli was toxic to cells, LOS from C. trachomatis did not induce any appreciable cell death, suggesting that it does not activate pyroptosis. Chlamydial LOS was also a poor stimulator of maturation of bone marrow-derived dendritic cells compared to E. coli LPS. Previous work from our group indicated that LOS synthesis during infection was necessary to alter host cell antigen presentation. However, direct delivery of LOS to cells in the absence of infection did not alter antigenic peptide presentation. Taken together, these data suggest that chlamydial LOS, which is remarkably conserved across the genus Chlamydia , may act both directly and indirectly to allow the pathogen to evade the innate and adaptive immune responses of the host., (Copyright © 2020 American Society for Microbiology.)

The obligate intracellular bacterium Chlamydia trachomatis causes the most prevalent bacterial sexually transmitted infection worldwide. CD4 T cells play a central role in the protective immunity against Chlamydia female reproductive tract (FRT) infection, while B cells are thought to be dispensable for resolution of primary Chlamydia infection in mouse models. We recently reported an unexpected requirement of B cells in local Chlamydia-specific CD4 T-cell priming and bacterial containment within the FRT. Here, we sought to tackle the precise effector function of B cells during Chlamydia primary infection. Using mixed bone marrow chimeras that lack B-cell-dependent Ag presentation (MHCII B - / - ) or devoid of circulating antibodies (AID -/- × μS -/- ), we show that Chlamydia-specific CD4 T-cell expansion does not rely on Ag presentation by B cells. Importantly, we demonstrate that antibody, but not B-cell-dependent Ag presentation, is required for preventing systemic bacterial dissemination following Chlamydia FRT infection., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chlamydia trachomatis has evolved strategies to prevent host cell apoptosis to evade the host immune defense. However, the precise mechanisms of antiapoptotic activity of C. trachomatis still need to be clarified. Pgp3, one of eight plasmid proteins of C. trachomatis, has been identified to be closely associated with chlamydial virulence. In this study, we attempted to explore the effects and the mechanisms of Pgp3 protein on apoptosis in HeLa cells; the results showed that Pgp3 increased Bcl-2/Bax ratio and prevented caspase-3 activation to suppress apoptosis induced by TNF-α and cycloheximide (CHX) through ERK1/2 pathway activation. Downregulation of DJ-1 with siRNA-DJ-1(si-DJ-1) reduced ERK1/2 phosphorylation and elevated apoptotic rate significantly in Pgp3-HeLa cells. However, inhibition of ERK1/2 signal pathway with ERK inhibitor PD98059 had little effect on DJ-1 expression. These findings confirm that plasmid protein Pgp3 contributes to apoptosis resistance through ERK1/2 signal pathway mediated by upregulation of DJ-1 expression. Therefore, the present study provided novel insights into the role of Pgp3 in apoptosis and suggested that manipulation of the host apoptosis response could be a new approach for the prevention and treatment of C. trachomatis infection., (© FEMS 2020.)

Background: Sirenomelia is a lethal congenital anomaly, presenting with fusion of lower extremities and malformed perineum. The pathogenesis is unclear, and "defective blastogenesis" is the proposed mechanism. Chlamydia trachomatis (CT) is an obligate intracellular pathogen which reportedly invades placenta and may result in fetal demise. It has documented cytopathogenic effects, specifically, cellular disruption, tissue dysgenesis, and genomic instability. Case report: An infant with sirenomelia was born as a product of 30 weeks of pregnancy, which was normal except for a persistent maternal CT infection. The infant expired shortly after birth. Conclusion: Fetal invasion by CT, conceivably, may induce structural anomalies, such as sirenomelia by virtue of its cytopathic effects. We intend to draw attention to such a possibility by reporting this case. This association, however, is speculative and more cases of sirenomelia with CT positive mothers need to be described in order to make definite conclusions about such a relationship.

The antibiotic, fosmidomycin (FSM) targets the methylerythritol phosphate (MEP) pathway of isoprenoid synthesis by inhibiting the essential enzyme, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) and is lethal to intracellular parasites and bacteria. The obligate intracellular bacterial pathogen, Chlamydia trachomatis, alternates between two developmental forms: the extracellular, infectious elementary body (EB), and the intracellular, replicative form called the reticulate body (RB). Several stressful growth conditions including iron deprivation halt chlamydial cell division and cause development of a morphologically enlarged, but viable form termed an aberrant body (AB). This phenotype constitutes the chlamydial developmental state known as persistence. This state is reversible as removal of the stressor allows the chlamydiae to re-enter and complete the normal developmental cycle. Bioinformatic analysis indicates that C. trachomatis encodes a homolog of Dxr, but its function and the requirement for isoprenoid synthesis in chlamydial development is not fully understood. We hypothesized that chlamydial Dxr (DxrCT) is functional and that the methylerythritol phosphate (MEP) pathway is required for normal chlamydial development. Thus, FSM exposure should be lethal to C. trachomatis. Overexpression of chlamydial Dxr (DxrCT) in Escherichia coli under FSM exposure and in a conditionally lethal dxr mutant demonstrated that DxrCT functions similarly to E. coli Dxr. When Chlamydia-infected cultures were exposed to FSM, EB production was significantly reduced. However, titer recovery assays, electron microscopy, and peptidoglycan labeling revealed that FSM inhibition of isoprenoid synthesis is not lethal to C. trachomatis, but instead induces persistence. Bactoprenol is a critical isoprenoid required for peptidoglycan precursor assembly. We therefore conclude that FSM induces persistence in Chlamydia by preventing bactoprenol production necessary for peptidoglycan precursor assembly and subsequent cell division., Competing Interests: The authors have declared that no competing interests exist.

Study Question: Can Chlamydia be found in the testes of infertile men?, Summary Answer: Chlamydia can be found in 16.7% of fresh testicular biopsies and 45.3% of fixed testicular biopsies taken from a selection of infertile men., What Is Known Already: Male chlamydial infection has been understudied despite male and female infections occurring at similar rates. This is particularly true of asymptomatic infections, which occur in 50% of cases. Chlamydial infection has also been associated with increased sperm DNA damage and reduced male fertility., Study Design, Size, Duration: We collected diagnostic (fixed, n = 100) and therapeutic (fresh, n = 18) human testicular biopsies during sperm recovery procedures from moderately to severely infertile men in a cross-sectional approach to sampling., Participants/materials, Setting, Methods: The diagnostic and therapeutic biopsies were tested for Chlamydia-specific DNA and protein, using real-time PCR and immunohistochemical approaches, respectively. Serum samples matched to the fresh biopsies were also assayed for the presence of Chlamydia-specific antibodies using immunoblotting techniques., Main Results and the Role of Chance: Chlamydial major outer membrane protein was detected in fixed biopsies at a rate of 45.3%. This was confirmed by detection of chlamydial DNA and TC0500 protein (replication marker). C. trachomatis DNA was detected in fresh biopsies at a rate of 16.7%, and the sera from each of these three positive patients contained C. trachomatis-specific antibodies. Overall, C. trachomatis-specific antibodies were detected in 72.2% of the serum samples from the patients providing fresh biopsies, although none of the patients were symptomatic nor had they reported a previous sexually transmitted infection diagnosis including Chlamydia., Limitations, Reasons for Caution: No reproductively healthy male testicular biopsies were tested for the presence of Chlamydia DNA or proteins or Chlamydia-specific antibodies due to the unavailability of these samples., Wider Implications for the Findings: Application of Chlamydia-specific PCR and immunohistochemistry in this human male infertility context of testicular biopsies reveals evidence of a high prevalence of previously unrecognised infection, which may potentially have a pathogenic role in spermatogenic failure., Study Funding/competing Interest(s): Funding for this project was provided by the Australian NHMRC under project grant number APP1062198. We also acknowledge assistance from the Monash IVF Group and Queensland Fertility Group in the collection of fresh biopsies, and the Monash Health and co-author McLachlan (declared equity interest) in retrieval and sectioning of fixed biopsies. E.M. declares an equity interest in the study due to financing of fixed biopsy sectioning. All other authors declare no conflicts of interest., Trial Registration Number: N/A., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen and the leading cause of preventable blindness in the developing world. C. trachomatis invades the epithelium of the conjunctiva and genital tract and replicates within an intracellular membrane-bound compartment termed the inclusion. To invade and replicate in mammalian cells, Chlamydia remodels epithelial surfaces by reorganizing the cytoskeleton and cell-cell adhesions, reprograms membrane trafficking, and modulates cell signaling to dampen innate immune responses. If the infection ascends to the upper female genital tract, it can result in pelvic inflammatory disease and tissue scarring. C. trachomatis infections are associated with infertility, ectopic pregnancies, the fibrotic disorder endometriosis, and potentially cancers of the cervix and uterus. Unfortunately, the molecular mechanisms by which this clinically important human pathogen subverts host cellular functions and causes disease have remained relatively poorly understood because of the dearth of molecular genetic tools to study Chlamydiae and limitations of both in vivo and in vitro infection models. In this review, we discuss recent advances in the experimental molecular tool kit available to dissect C. trachomatis infections with a special focus on Chlamydia -induced epithelial barrier disruption by regulating the structure, function, and dynamics of epithelial cell-cell junctions., Competing Interests: Competing interests: RHV is a member of the scientific advisory board of Meridian Biosciences. LD declares that he has no competing interests.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

The sexually transmitted pathogen Chlamydia trachomatis (CT) is able to replicate and survive in human intestinal epithelial cells, being the gastro-intestinal tract a suitable site of residence for this microorganism. In this context, no detailed information about the mechanisms of cell death in intestinal cell lines after a chlamydial infection is available. The aim of this study was to compare the effect of two different CT serovars (D and L2) on the survival/death of different intestinal cell lines (Caco-2 and COLO-205), using endocervical cells (HeLa) as a reference model of genital infection. Seventy two hours after chlamydial infection at different multiplicity of infection (MOI) levels, the viability of HeLa, Caco-2 and COLO 205 cells was evaluated through dose-response experiments by means of a MTS-based assay. To get deeper insights in the mechanisms of cell death induced by CT, cell viability was assessed in presence of different inhibitors (i.e. pan-caspase inhibitor Z-VAD, necroptosis inhibitor Necrostatin-1, hydrogen peroxide scavenger catalase, caspase-1 inhibitor Ac-YVAD-cmk). Moreover, the activation of effector caspases and the presence of cellular apoptotic/necrotic changes were evaluated at different time points after CT infection. Our results demonstrated that, for both chlamydial serovars, intestinal cell lines are more resistant to CT-induced cell death compared to HeLa, thus representing a suitable 'niche' for chlamydial residence and replication. In literature, apoptosis has been widely described to be the main cell death mechanism elicited by chlamydia infection. However, our data demonstrate that necroptosis plays a relevant role, proceeding in parallel with apoptosis. The protective effect of catalase suggests the involvement of oxidative stress in triggering both cell death pathways. Moreover, we demonstrated that caspase-1 is involved in CT-induced cell death, potentially contributing to host inflammatory response and tissue damage. Cells infected by L2 serovar displayed a higher activation of effector caspases compared to cells infected with serovar D, suggesting a serovar-specific activation of apoptotic pathways and potentially explaining the greater virulence of L serovars. Finally, we found that Chlamydia elicits the early externalization of phosphatidylserine on the external leaflet of plasma membrane independently of caspase activation., Competing Interests: The authors have declared that no competing interests exist.

Chlamydia trachomatis is a bacterial pathogen causing ocular and genital infections in humans. C. trachomatis multiplies exclusively inside host cells within a characteristic vacuole, from where it manipulates host cells by injecting them with type III secretion effector proteins. Here, we identified CteG as the first C. trachomatis effector associated with the Golgi. For this, C. trachomatis strains expressing candidate effectors fused to a double hemagglutinin (2HA) tag were constructed. Then, among these strains, immunofluorescence microscopy revealed that CteG-2HA was delivered into the cytoplasm of infected cells. Between 16-20 h post-infection, CteG-2HA mostly associated with the Golgi; however, CteG-2HA also appeared at the host cell plasma membrane, and at 30 or 40 h post-infection this was its predominant localization. This change in the main localization of CteG-2HA was independent of intact microfilaments or microtubules. Ectopic expression of different regions of CteG (656 amino acid residues) in uninfected cells revealed that its first 100 residues contain a Golgi targeting region. Although a C. trachomatis cteG mutant did not display a defect in intracellular multiplication, CteG induced a vacuolar protein sorting defect when expressed in Saccharomyces cerevisiae. This suggested that CteG might function by subverting host cell vesicular transport.

Chlamydial infection requires the formation of a membrane-bound vacuole, termed the inclusion, that undergoes extensive interactions with select host organelles. The importance of the Inc protein CT229 in the formation and maintenance of the chlamydial inclusion was recently highlighted by studies demonstrating that its absence during infection results in reduced bacterial replication, premature inclusion lysis, and host cell death. Previous reports have indicated that CT229 binds Rab GTPases; however, the physiological implications of this interaction are unknown. Here, we show that CT229 regulates host multivesicular trafficking by recruiting multiple Rab GTPases and their cognate effectors to the inclusion. We demonstrate that CT229 specifically modulates clathrin-coated vesicle trafficking and regulates the trafficking of transferrin and the mannose-6-phosphate receptor, both of which are crucial for proper chlamydial development. This study highlights CT229 as a master regulator of multiple host vesicular trafficking pathways essential for chlamydial infection., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sulforaphane (SFN), a phytochemical found in broccoli and other cruciferous vegetables, is a potent antioxidant and anti-inflammatory agent with reported effects in cancer chemoprevention and suppression of infection with intracellular pathogens. Here we report on the impact of SFN on infection with Chlamydia trachomatis (Ct), a common sexually transmitted pathogen responsible for 131 million new cases annually worldwide. Astoundingly, we find that SFN as well as broccoli sprouts extract (BSE) promote Ct infection of human host cells. Both the number and size of Ct inclusions were increased when host cells were pretreated with SFN or BSE. The initial investigations presented here point to both the antioxidant and thiol alkylating properties of SFN as regulators of Ct infection. SFN decreased mitochondrial protein sulfenylation and promoted Ct development, which were both reversed by treatment with mitochondria-targeted paraquat (MitoPQ). Inhibition of the complement component 3 (complement C3) by SFN was also identified as a mechanism by which SFN promotes Ct infections. Mass spectrometry analysis found alkylation of cysteine 1010 (Cys1010) in complement C3 by SFN. The studies reported here raise awareness of the Ct infection promoting activity of SFN, and also identify potential mechanisms underlying this activity., (© 2018 The Protein Society.)

In the study of intracellular bacteria that reside within a membrane-bound vacuole, there are many questions related to how prokaryotic or eukaryotic transmembrane or membrane-associated proteins are organized and function within the membranes of these pathogen-containing vacuoles. Yet this host-pathogen interaction interface has proven difficult to experimentally resolve. For example, one method to begin to understand protein function is to determine the protein-binding partners; however, examining protein-protein interactions of hydrophobic transmembrane proteins is not widely successful using standard immunoprecipitation or coimmunoprecipitation techniques. In these scenarios, the lysis conditions that maintain protein-protein interactions are not compatible with solubilizing hydrophobic membrane proteins. In this chapter, we outline two proximity labeling systems to circumvent these issues to study (1) eukaryotic proteins that localize to the membrane-bound inclusion formed by Chlamydia trachomatis using BioID, and (2) chlamydial proteins that are inserted into the inclusion membrane using APEX2. BioID is a promiscuous biotin ligase to tag proximal proteins with biotin. APEX2 is an ascorbate peroxidase that creates biotin-phenoxyl radicals to label proximal proteins with biotin or 3,3'-diaminobenzidine intermediates for examination of APEX2 labeling of subcellular structures using transmission electron microscopy. We present how these methods were originally conceptualized and developed, so that the user can understand the strengths and limitations of each proximity labeling system. We discuss important considerations regarding experimental design, which include careful consideration of background conditions and statistical analysis of mass spectrometry results. When applied in the appropriate context with adequate controls, these methods can be powerful tools toward understanding membrane interfaces between intracellular pathogens and their hosts.

Other than its routine application for capturing pure cell populations from fixed tissue sections for diverse downstream molecular assays, laser microdissection enables isolation of single live cells. Here we describe a method for the isolation of single Chlamydia trachomatis-infected cells using a laser microdissection system, in which the dissected samples are captured via gravity. Cells infected by C. trachomatis at low multiplicity of infection are marked with the fluorescent Golgi-specific probe BODIPY ® FL C5-ceramide, to facilitate identification of the cells with chlamydial inclusions under the microscope. Individual C. trachomatis-infected cells are harvested into separate wells with a pregrown host cell monolayer. Inclusions in harvested cells maturate, and the released elementary bodies infect the host cell monolayer, thus initiating propagation of single inclusion-derived Chlamydia. The method can be used for generation of microbiological clones of C. trachomatis and recovery of transformants and mutants. Isolated single Chlamydia-infected cells can also be examined by diverse downstream molecular assays to reveal unknown features of the Chlamydia replication at a single inclusion level.

Chlamydia trachomatis is an obligate intracellular bacterium that scavenges host metabolic products for its replication. Mitochondria are the power plants of eukaryotic cells and provide most of the cellular ATP via oxidative phosphorylation. Several intracellular pathogens target mitochondria as part of their obligatory cellular reprogramming. This study was designed to analyse the mitochondrial morphological changes in response to C. trachomatis infection in HeLa cells. Mitochondrial elongation and fragmentation were found at the early stages and late stages of C. trachomatis infection, respectively. C. trachomatis infection-induced mitochondrial elongation was associated with the increase of mitochondrial respiratory activity, ATP production, and intracellular growth of C. trachomatis. Silencing mitochondrial fusion mediator proteins abrogated the C. trachomatis infection-induced elevation in the oxygen consumption rate and attenuated chlamydial proliferation. Mechanistically, C. trachomatis induced the elevation of intracellular cAMP at the early phase of infection, followed by the phosphorylation of fission-inactive serine residue 637 (S637) of Drp1, resulting in mitochondrial elongation. Accordingly, treatment with adenylate cyclase inhibitor diminished mitochondrial elongation and bacterial growth in infected cells. Collectively, these results strongly indicate that C. trachomatis promotes its intracellular growth by targeting mitochondrial dynamics to regulate ATP synthesis via inhibition of the fission mediator Drp1., (© 2018 John Wiley & Sons Ltd.)

Chlamydia trachomatis (Ct) infections can cause bacterial sexually-transmitted and preventable blindness. The Ct infections induced excessive cytokines generation which attributed to pathologic changes in host cells. However, the precise mechanisms of Ct-induced cytokines production are still unclear.CT143 protein was identified as a novel Ct specific protein with high immunogenicity. In the present study. The CT143 fusion protein was recombined and purified. The mice immune serum was prepared by immunizing BALB/c mice with the purified fusion protein. The specificity of the antibody was confirmed using Immunoblotting. Indirect immunoflurescence assay (IFA) and Immunoblotting assays were performed to detect the temporal and spatial characteristics of CT143 in Ct infected cells. ELISA was performed to analyze the secretion of proinflammatory cytokines IL-1β, IL-8 and TNF-α by human macrophages under the stimulation of CT143 protein. Finally, the involvement of p38 signaling in CT143-induced cytokine secretion was validated. CT143 protein was located in the inclusion body and represented an Elementary body (EB)-related protein, which may be encoded by the mid- and late-stage expressing genes. CT143 protein could stimulate the secretion of inflammatory cytokines in macrophages which differentiated from THP-1 This induction may be mediated by the activation of p38 signaling. In summary, CT143 protein is involved in inflammatory processes during Ct infection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 .

Chlamydia belong to the group of obligate intracellular bacteria that reside in a membrane bound vacuole during the entire intracellular phase of their life cycle. This vacuole called inclusion shields the bacteria from adverse influences in the cytosol of the host cell like the destructive machinery of the cell-autonomous defence system. The inclusion thereby prevents the digestion and eradication in specialised compartments of the intact and viable cell called phagolysosomes or autophagolysosomes. It is becoming more and more evident that keeping the inclusion intact also prevents the onset of cell intrinsic cell death programmes that are activated upon damage of the inclusion and direct the cell to destruct itself and the pathogen inside. Chlamydia secrete numerous proteins into the inclusion membrane to protect and stabilise their unique niche inside the host cell. We will focus in this review on the diverse attack strategies of the host aiming at the destruction of the Chlamydia-containing inclusion and will summarise the current knowledge on the protection mechanisms elaborated by the bacteria to maintain the integrity of their replication niche., (© 2018 John Wiley & Sons Ltd.)

Chlamydia trachomatis (Ctr) accounts for >130 million human infections annually. Since chronic Ctr infections are extremely difficult to treat, there is an urgent need for more effective therapeutics. As an obligate intracellular bacterium, Ctr strictly depends on the functional contribution of the host cell. Here, we combined a human genome-wide RNA interference screen with metabolic profiling to obtain detailed understanding of changes in the infected cell and identify druggable pathways essential for Ctr growth. We demonstrate that Ctr shifts the host metabolism toward aerobic glycolysis, consistent with increased biomass requirement. We identify key regulator complexes of glucose and nucleotide metabolism that govern Ctr infection processes. Pharmacological targeting of inosine-5'-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in guanine nucleotide biosynthesis, efficiently inhibits Ctr growth both in vitro and in vivo. These results highlight the potency of genome-scale functional screening for the discovery of drug targets against bacterial infections., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Chlamydia trachomatis is a widespread sexually transmitted pathogen that resides within a special vacuole inside host cells. Although acute infection can be treated with antibiotics, chlamydia can enter persistent state, leading to chronic infection that is difficult to cure. Thus, novel anti-chlamydial compounds active against persistent chlamydia are required. Chlamydiae rely upon type III secretion system (T3SS) to inject effector proteins into host cell cytoplasm, and T3SS inhibitors are viewed as promising compounds for treatment of chlamydial infections. C. trachomatis ATPase SctN is an important T3SS component and has not been targeted before. We thus used virtual screening against homology modeled SctN structure to search for SctN inhibitors. Selected compounds were tested for their ability to inhibit chlamydial survival and development within eukaryotic cells, and for the ability to suppress normal T3SS functioning. We identified two compounds that were able to block normal protein translocation through T3SS and inhibit chlamydial survival within eukaryotic cells in 50-100 μm concentrations. These two novel T3SS inhibitors also possessed relatively low toxicity toward eukaryotic cells. A small series of derivatives was further synthesized for the most active of two inhibitors to probe SAR properties., (© 2017 John Wiley & Sons A/S.)

Infection with Chlamydia trachomatis drives severe mucosal immunopathology; however, the immune responses that are required for mediating pathology vs. protection are not well understood. Here, we employed a mouse model to identify immune responses required for C. trachomatis -induced upper genital tract pathology and to determine whether these responses are also required for bacterial clearance. In mice as in humans, immunopathology was characterized by extravasation of leukocytes into the upper genital tract that occluded luminal spaces in the uterus and ovaries. Flow cytometry identified these cells as neutrophils at early time points and CD4 + and CD8 + T cells at later time points. To determine what draws these cells to C. trachomatis -infected tissue, we measured the expression of 700 inflammation-related genes in the upper genital tract and found an up-regulation of many chemokines, including a node of interaction between CXCL9/10/11 and their common receptor CXCR3. Either depleting neutrophils or reducing T-cell numbers by CXCR3 blockade was sufficient to significantly ameliorate immunopathology but had no effect on bacterial burden, demonstrating that these responses are necessary for mucosal pathology but dispensable for C. trachomatis clearance. Therapies that specifically target these host responses may therefore prove useful in ameliorating C. trachomatis -induced pathology without exacerbating infection or transmission., Competing Interests: The authors declare no conflict of interest.

Background: Understanding the relationship between the levonorgestrel (LNG)-releasing intrauterine system (IUS) and sexually transmitted infections (STIs) is increasingly important as use of the LNG-IUS grows to include women at higher risk for STIs. This study assessed the impact of the LNG-IUS on development of Chlamydia trachomatis pelvic inflammatory disease, using a baboon model., Methods: Baboons with and those without the LNG-IUS were cervically inoculated with C. trachomatis and monitored daily, and cervical and fallopian tube swab specimens were collected weekly for C. trachomatis quantitation by nucleic acid amplification testing and culture. Vaginal swab specimens were collected for cytokine analysis, and serum samples were obtained for detection of C. trachomatis antibodies., Results: The LNG-IUS resulted in an increased C. trachomatis burden in the cervix, with the bacterial burden in the LNG-IUS group diverging from that in the non-LNG-IUS group by 6 weeks after infection. One of 7 baboons in the non-LNG-IUS group and 2 of 6 in the LNG-IUS group developed pelvic inflammatory disease, while 3 animals in each group met criteria suggestive of pelvic inflammatory disease. LNG-IUS increased baseline interleukin 8 levels but failed to further upregulate interleukin 8 during infection. In LNG-IUS recipients, early perturbations in the interleukin 1β axis corresponded to decreased C. trachomatis clearance and increased T-helper type 2 immune responses., Conclusion: LNG-IUS use results in delayed clearance of C. trachomatis and might alter the reproductive tract immune environment., (© The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Chlamydia are gram-negative obligate intracellular bacteria that replicate within a discrete cellular vacuole, called an inclusion. Although it is known that Chlamydia require essential nutrients from host cells to support their intracellular growth, the molecular mechanisms for acquiring these macromolecules remain uncharacterized. In the present study, it was found that the expression of mammalian cell glucose transporter proteins 1 (GLUT1) and glucose transporter proteins 3 (GLUT3) were up-regulated during chlamydial infection. Up-regulation was dependent on bacterial protein synthesis and Chlamydia-induced MAPK kinase activation. GLUT1, but not GLUT3, was observed in close proximity to the inclusion membrane throughout the chlamydial developmental cycle. The proximity of GLUT1 to the inclusion was dependent on a brefeldin A-sensitive pathway. Knockdown of GLUT1 and GLUT3 with specific siRNA significantly impaired chlamydial development and infectivity. It was discovered that the GLUT1 protein was stabilized during infection by inhibition of host-dependent ubiquitination of GLUT1, and this effect was associated with the chlamydial deubiquitinase effector protein CT868. This report demonstrates that Chlamydia exploits host-derived transporter proteins altering their expression, turnover and localization. Consequently, host cell transporter proteins are manipulated during infection as a transport system to fulfill the carbon source requirements for Chlamydia., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chlamydia antigen analysis enables understanding of disease pathogenesis, facilitates development of diagnostic immunoassays and is essential to the design of a subunit Chlamydia trachomatis vaccine. Using an autobiographical narrative, I review over three decades of antigen analysis research findings coming from my research laboratory and provide an outlook to the broader field of Chlamydia seroepidemiology and vaccinology. Based on the experiences of my scientific career I conclude with thoughts for young scientists newly entering the Chlamydia research field., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Development of approaches to genetically manipulate Chlamydia is fostering important advances in understanding pathogenesis. F luorescence- r eported a llelic e xchange m utagenesis (FRAEM) now enables the complete deletion of specific genes in C. trachomatis L2. We have leveraged this technology to delete the coding sequences for a known type III effector. The evidence provided here indicates that CT694/CTL0063 is a virulence protein involved in chlamydial invasion. Based on our findings, we designate the gene product corresponding to ct694-ctl0063t ranslocated m embrane-associated e ffector A (TmeA). Deletion of tmeA did not impact development of intracellular chlamydiae. However, the absence of TmeA manifested as a decrease in infectivity in both tissue culture and murine infection models. The in vitro defect was reflected by impaired invasion of host cells. TmeA binds human AHNAK, and we demonstrate here that AHNAK is transiently recruited by invading chlamydiae. TmeA, however, is not required for endogenous AHNAK recruitment. TmeA also impairs AHNAK-dependent actin bundling activity. This TmeA-mediated effect likely does not explain impaired invasion displayed by the tmeA strain of Chlamydia , since AHNAK-deficient cells revealed no invasion phenotype. Overall, our data indicate the efficacy of FRAEM and reveal a role of TmeA during chlamydial invasion that manifests independently of effects on AHNAK., (Copyright © 2017 American Society for Microbiology.)

Background: Rectal STI coinfection models enhance the understanding of rectal HIV transmission risk factors., Materials and Methods: Rhesus macaques (n=9) were exposed to one of three rectal Chlamydia trachomatis (CT) challenges: C. trachomatis L 2 (CT-L 2 ); C. trachomatis serovar E (CT-E), followed by CT-L 2 ; or CT-E, treatment/clearance, then CT-L 2 . Infections were monitored by PCR. Weekly blood and rectal secretion/lavage samples were collected for cytokine analyzes and/or epithelial sloughing, occult, and overt blood determinations., Results: Chlamydial infections were successfully established in each animal, with varying degrees of persistence. Mucosal IL-1beta was upregulated in animals consecutively infected with CT-E then CT-L 2 (P=.05). Epithelial sloughing was also significantly increased post-infection in this group (P=.0003)., Conclusions: This study demonstrates successful rectal infection of rhesus macaques with CT-E and CT-L 2 and describes measures of assessing rectal inflammation and pathology. Different infection strategies yield varying inflammatory and pathologic outcomes, providing well-described models for future SIV/SHIV susceptibility studies., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A reliable overview of data on the prevalence of Chlamydia trachomatis (CT) in Russia is lacking and needed. All the available data on CT prevalence were analyzed in a systematic literature review on CT prevalence in Russia, strengthened with data from the multicenter study among 1263 people in the second-largest Russian megalopolis, St. Petersburg, testing for CT DNA in urethral, anal, cervical and prostate samples. A total of 10 articles met the inclusion criteria. The overall average prevalence of genital CT infections in Russian populations ranged from 2.9% to 33%. Risk factors included being symptomatic (P = 0.004; in men P < 0.001), being younger than 30 years (P = 0.001) and being a man who has sex with men (MSM) (P = 0.0084). Main limitations included the lack of studies in MSM. CT prevalence was higher in the groups where urethral and prostate secretion samples were pooled (5.2%-7.3% vs 3.2% in the urethra only). The data on CT prevalence in a range of Russian populations are analyzed and reported. Prostate secretions represent an additional sampling material for the study of CT infection in men. CT detection in some settings in St. Petersburg yielded levels of reliability comparable with internationally available tests. The initiation of screening programs for Chlamydia infections in Russia should be considered., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Chlamydia trachomatis is the etiological agent for the most prevalent bacterial sexually transmitted infection in both developed and developing countries. The aim of present study was to characterize the antibody response between two groups of individuals, having either a single C. trachomatis infection and or repeated infections., Material and Methods: Current study consisted of two groups, one with an initial Chlamydia infection and a second with repeated infections. A titre based estimation of specific serum (IgG and IgA) levels using ELISA were performed, which further validated by western blot. In vitro neutralizing ability of each patient's serum against both homologous and heterologous strains was also determined., Results: Individuals infected with one of the C. trachomatis serovars D, E or K exhibited a strong systemic antibody response as characterized by ELISA and western blot. These individuals may have developed at least some level of protection as they only represented single infection. By comparison, individuals infected with serovar D, E or F that exhibited low systemic antibody response often presented repeated C. trachomatis infections, suggesting an association with poor immune response. An in vitro neutralizing level of 60-90% was observed in the human sera against homologous serovar D and two heterologous C. trachomatis serovars E and K, compared to <40% against heterologous serovars F., Conclusion: Individuals infected with serovars D and K showed a potential association between circulating antibody response and re-infection risk. While the patients infected with serovars E showed a disconnection between systemic antibody response and re-infection risk.

Objective: This study included women attending primary health care units in Botucatu, São Paulo, Brazil, to assess the cervicovaginal levels of human β-defensin (hBD) 1, 2, 3, and 4 during Chlamydia trachomatis infection., Patients and Methods: Cervicovaginal samples were collected for Pap testing and assessing the presence of infection by C. trachomatis, human papillomavirus, Neisseria gonorrhoeae, and Trichomonas vaginalis. Vaginal smears were taken to evaluate local microbiota. Human β-defensin levels were determined using enzyme-linked immunosorbent assay in cervicovaginal fluid samples. Seventy-four women with normal vaginal microbiota and no evidence of infection were included in hBD quantification assays; 37 tested positive for C. trachomatis and 37 were negative. Statistical analysis was performed using Mann-Whitney U test., Results: Women positive for C. trachomatis had significantly lower cervicovaginal hBD-1, hBD-2, and hBD-3 compared with those who tested negative (hBD-1: 0 pg/mL [0-2.1] vs 1.6 pg/mL [0-2.4], p < .0001; hBD-2: 0 pg/mL [0-3.9] vs 0.61 pg/mL [0-8.9], p = .0097; and hBD-3: 0 pg/mL [0-4.3] vs 0.28 pg/mL [0-8.4], p = .0076). Human β-defensin 4 was not detected., Conclusions: Lower levels of hBD-1, hBD-2, and hBD-3 in cervicovaginal fluid were detected in the presence of C. trachomatis infection.

We studied infection and immunity of hysterectomized mice infected with Chlamydia muridarum and Chlamydia trachomatis to determine if there were differences between these species in their ability to infect vaginal squamous epithelial cells in vivo independently of proximal upper genital tract tissues. We found that C. muridarum readily colonized and infected vaginal squamous epithelial cells, whereas C. trachomatis did not. Primary infection of the vaginal epithelium with C. muridarum produced infections of a duration longer than that reported for normal mice. Infection resulted in an inflammatory response in the vagina characterized by neutrophils and infiltrating submucosal plasma cells consisting primarily of T cells. Despite the delayed clearance, rechallenged C. muridarum -infected mice were highly immune. Mice vaginally infected with C. muridarum produced serum and vaginal wash antibodies and an antigen-specific gamma interferon-dominated Th1-biased T cell response. By comparison, mice vaginally infected with C. trachomatis exhibited transient low-burden infections, produced no detectable tissue inflammatory response, and failed to seroconvert. We discuss how these marked differences in the biology of vaginal infection between these otherwise genetically similar species are possibly linked to pathogen-specific virulence genes and how they may influence pathology and immunity in the upper genital tract., (Copyright © 2017 American Society for Microbiology.)

Miscarriage affects ~20% of pregnancies and maternal infections account for ~15% of early miscarriages. Chlamydia trachomatis (Ct) has been associated with miscarriage but the underlying mechanisms are unknown. Successful implantation requires endometrial stromal cell (ESC) decidualisation. Maintenance of pregnancy requires angiogenesis, establishment of the correct cellular milieu and trophoblast invasion, all of which involve the action of chemokines. Our objective was to determine whether Ct infection impacts upon ESC decidualisation and chemokine secretion. Human primary ESC were decidualised in-vitro, infected with Ct serovar E, and changes in expression of genes of interest were measured using RT-PCR, proteomic array and ELISA. We demonstrate for the first time that Ct can infect and proliferate in ESC. Expression of the decidualisation marker prolactin was decreased in Ct-infected ESC at both mRNA and protein levels. Ct infection altered the chemokine profile of decidualised ESC as shown by proteomic array. Chemokines CXCL12 and CXCL16, important for trophoblast invasion, were analysed further and expression was reduced in infected decidualised cells at mRNA and protein levels. Our data indicate that Ct infection of ESC impairs decidualisation and alters chemokine release. These findings at least partially explain how Ct infection could result in adverse pregnancy outcomes.

Chlamydia trachomatis is the most prevalent infection of the genital tract in women worldwide. C. trachomatis has a tendency to cause persistent infection and induce a state of chronic inflammation, which has been reported to play a role in carcinogenesis. We report that persistent C. trachomatis infection increases the expression of inflammatory tumour cytokines and upregulates molecular biomarkers such as podoplanin, Wilms' tumour gene 1 and osteopontin in primary cultures of mesothelial cells (Mes1) and human mesothelioma cells (NCI). Infection experiments showed that Mes1 and NCI supported the growth of C. trachomatisin vitro, and at an m.o.i. of 4, the inclusion-forming units/cell showed many intracellular inclusion bodies after 3 days of infection. However, after 7 days of incubation, increased proliferative and invasive activity was also observed in Mes1 cells, which was more evident after 14 days of incubation. ELISA analysis revealed an increase in vascular endothelial growth factor, IL-6, IL-8, and TNF-α release in Mes1 cells infected for a longer period (14 days). Finally, real-time PCR analysis revealed a strong induction of podoplanin, Wilms' tumour gene 1 and osteopontin gene expression in infected Mes1 cells. The aim of the present study was to investigate the inflammatory response elicited by C. trachomatis persistent infection and the role played by inflammation in cell proliferation, secretion of proinflammatory cytokines and molecular biomarkers of cancer. The results of this study suggest that increased molecular biomarkers of cancer by persistent inflammation from C. trachomatis infection might support cellular transformation, thus increasing the risk of cancer.

Sexually transmitted infections are an important cause of morbidity among sexually active women worldwide, and have been implicated as cofactors in the pathogenesis of cervical cancer. We investigated the prevalence of human papillomavirus (HPV), Chlamydia trachomatis (CT), and Trichomonas vaginalis (TV), and accessed the diversity of HPV in women with normal and abnormal cytology in Manaus, Brazil. We used polymerase chain reaction and HPV genotyping by direct sequencing. The chi-square test was used to calculate the absolute and relative frequencies of the categorical variables, and Fisher's test was used when P < 0.05. The level of significance was set at 5%. All statistical analyses were performed using R 2.9.0. There were statistically significant differences in age (P = 0.0395), education level (P = 0.0131), sexual partners (P = 0.0211), condom use (P = 0.0039), marital status (P < 0.0001), and pregnancy (P = 0.0003) between the normal and abnormal groups. HPV DNA was found in 36.56 and 93.88% of subjects in the normal and abnormal groups, respectively. A total of 19 genotypes were detected; HPV16 was the most common, followed by HPV58. The percentages of TV and CT DNA were 18.04 and 9.02% in the normal group, respectively. The percentages of HPV/TV and HPV/CT coinfection were 12.5% each in women with normal cytology. These findings improve our understanding of HPV, CT, and TV, and the distribution of HPV types, which may be relevant to vaccination strategies for protecting women from the north of Brazil from cervical cancers and precancerous lesions.

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection. However, whether Chlamydia trachomatis has a negative impact on sperm quality and male fertility is still controversial. Herein, we report the effects on sperm quality of the in vitro exposure of spermatozoa to Chlamydia trachomatis, and also the effects of male genital infection on male fertility using an animal model. Human and mouse sperm were obtained from healthy donors and cauda epididimys from C57BL/6 mice, respectively. Highly motile human or mouse spermatozoa were in vitro exposed to C. trachomatis (serovar E or LGV) or C. muridarum, respectively. Then, sperm quality parameters were analyzed. Moreover, male fertility of Chlamydia muridarum infected male C57BL/6 mice was assessed. Human or murine sperm in vitro exposed to increasing bacterial concentrations or soluble factors from C. trachomatis or C. muridarum, respectively, did not show differences in sperm motility and viability, apoptosis, mitochondrial membrane potential, DNA fragmentation, ROS production and lipid peroxidation levels, when compared with control sperm (p > 0.05). Moreover, no differences in fertility parameters (potency, fecundity, fertility index, pre- and post-implantation loss) were observed between control and infected males. In conclusion, our results indicate that Chlamydia spp. neither directly exerts deleterious effects on spermatozoa nor impairs male fertility.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections (STIs) and preventable blindness. Untreated, asymptomatic infection as well as frequent re-infection are common and may drive pelvic inflammatory disease, ectopic pregnancy, and infertility. In vivo models of chlamydial infection continue to be instrumental in progress toward a vaccine and further elucidating the pathogenesis of this intracellular bacterium, however significant gaps in our understanding remain. Chlamydial host cell exit occurs via two mechanisms, lysis and extrusion, although the latter has yet to be reported in vivo and its biological role is unclear. The objective of this study was to investigate whether chlamydial extrusions are shed in vivo following infection with multiple strains of Chlamydia . We utilized an established C3H/HeJ murine cervicovaginal infection model with C. trachomatis serovars D and L2 and the Chlamydia muridarum strain MoPn to monitor the (i) time course of infection and mode of host cell exit, (ii) mucosal and systemic immune response to infection, and (iii) gross and histopathology following clearance of active infection. The key finding herein is the first identification of chlamydial extrusions shed from host cells in an in vivo model. Extrusions, a recently appreciated mode of host cell exit and potential means of dissemination, had been previously observed solely in vitro . The results of this study demonstrate that chlamydial extrusions exist in vivo and thus warrant further investigation to determine their role in chlamydial pathogenesis.

Most Chlamydia species carry a 7.5kb plasmid encoding eight open reading frames conventionally called plasmid glycoproteins 1-8 or pGP1-8. Although the plasmid is not critical for chlamydial growth in vitro, its role in chlamydial pathogenesis is clearly demonstrated in the genital tracts of mice infected with Chlamydia muridarum, a model for investigating the human pathogen Chlamydia trachomatis. Plasmid-free C. trachomatis is also attenuated in both the mouse genital tract and nonhuman primate ocular tissue. Deficiency in pGP3 alone, which is regulated by pGP4, largely reproduced the in vivo but not in vitro phenotypes of the plasmid-free organisms, suggesting that pGP3 is a key in vivo virulence factor. The positive and negative regulations of some chromosomal genes by pGP4 and pGP5, respectively, may allow the plasmid to promote chlamydial adaptation to varied animal tissue environments. The focus of this review is to summarize the progress on the pathogenic functions of the plasmid-encoded open reading frames, which may motivate further investigation of the molecular mechanisms of chlamydial pathogenicity and development of medical utility of the chlamydial plasmid system., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We studied whether antibody to two chlamydial proteins (TroA and HtrA) could be used as biomarkers of Chlamydia trachomatis infection., Methods: Recombinant proteins C. trachomatis TroA and HtrA were used as antigens in enzyme immunoassay (EIA). Both IgG and IgA antibody responses were studied., Results: IgG or IgA antibody to either protein was infrequently detected in sera from healthy blood donors or virgin girls. Patients attending the STI Clinic and patients with perihepatitis had often IgG antibody against TroA (25 and 50 % respectively) and HtrA (21 and 38 % respectively). Especially in sera from patients with chlamydial perihepatitis, the A 450nm values with TroA were high (mean 1.591). A positive correlation between C. trachomatis MIF antibody and TroA (r  = 0.7) as well as HtrA (r  = 0.5) antibody was observed in sera from STI clinic patients and perihepatitis patients. Individuals with C. trachomatis infection and positive serology already when seeking medical attention had higher A 450nm values for TroA (0.638) and HtrA (0.836) than patients with no marker of previous exposure or with no infection (0.208 and 0.234 respectively). Diagnosis of genital C. trachomatis infection is often NAAT-based, whereas serology has little value in testing for uncomplicated genital C. trachomatis infection. TroA and HtrA antibodies are potential biomarkers for evaluation of ascending and repeated C. trachomatis infection.

The growing popularity of levonorgestrel (LNG)-releasing intra-uterine systems for long-acting reversible contraception provides strong impetus to define immunomodulatory properties of this exogenous progestin. In initial in vitro studies herein, we found LNG significantly impaired activation of human dendritic cell (DCs) and their capacity to promote allogeneic T cell proliferation. In follow-up studies in a murine model of intranasal Chlamydia trachomatis infection, we analogously found that LNG treatment prior to infection dramatically reduced CD40 expression in DCs isolated from draining lymph nodes at 2 days post infection (dpi). At 12 dpi, we also detected significantly fewer CD4 + and CD8 + T cells in the lungs of LNG-treated mice. This inhibition of DC activation and T cell expansion in LNG-treated mice also delayed chlamydial clearance and the resolution of pulmonary inflammation. Conversely, administering agonist anti-CD40 monoclonal antibody to LNG-treated mice at 1 dpi restored lung T cell numbers and chlamydial burden at 12 dpi to levels seen in infected controls. Together, these studies reveal that LNG suppresses DC activation and function, and inhibits formation of pathogen-specific T cell immunity. They also highlight the need for studies that define in vivo effects of LNG use on human host response to microbial pathogens.

Genital tract infections with ureaplasma urealyticum (UU) and chlamydia trachomatis (CT) are the most frequent sexually-transmitted disease worldwide. UU and CT infections are considered to be the leading cause for infertility and adverse pregnancy outcomes. However, little is known about the specific effect of cervical UU and CT infections on the etiology of female infertility, as well as the pregnancy outcomes of the patients undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET). In order to find the association between cervical UU and/or CT infection and pregnancy outcomes, we conducted a retrospective case-control study on the patients undergoing IVF/ICSI-ET with cervical UU and/or CT infection. A total of 2208 patients who received IVF/ICSI-ET were enrolled in this study. Data on the general conditions, pregnancy history and clinical pregnant outcomes were analyzed in terms of the cervical UU and CT detection. Our results revealed that cervical UU and CT infections were the risk factors for ectopic pregnancy and tubal factor-induced infertility. Moreover, the pregnancy rate, abortion rate, ectopic pregnancy rate and premature birth rate in patients with UU and/or CT infections showed no significant difference when compared with the control group. We recommend that cervical UU and CT detection should be an optional item for infertility patients and clinical UU detection should differentiate the subtypes of cervical UU. Positive cervical UU and CT infections should not be taken as strict contraindications for IVF/ICSI-ET.

While ascension of Chlamydia trachomatis into the upper genital tract of women can cause pelvic inflammatory disease and Fallopian tube damage, most infections elicit no symptoms or overt upper genital tract pathology. Consistent with this asymptomatic clinical presentation, genital C. trachomatis infection of women generates robust TH2 immunity. As an animal model that modeled this response would be invaluable for delineating bacterial pathogenesis and human host defenses, herein we explored if pathogen-specific TH2 immunity is similarly elicited by intravaginal (ivag) infection of mice with oculogenital C. trachomatis serovars. Analogous to clinical infection, ascension of primary C. trachomatis infection into the mouse upper genital tract produced no obvious tissue damage. Clearance of ivag challenge infection was mediated by interferon (IFN)-γ-producing CD4+ T cells, while IFN-γ signaling blockade concomitant with a single ivag challenge promoted tissue damage by enhancing Chlamydia-specific TH17 immunity. Likewise, IFN-γ and IL-17 signaling blockade or CD4+ T cell depletion eliminated the genital pathology produced in untreated controls by multiple ivag challenge infections. Conversely, we were unable to detect formation of pathogen-specific TH2 immunity in C. trachomatis-infected mice. Together, our work revealed C. trachomatis infection of mice generates TH1 and TH17 immune responses that promote pathogen clearance and immunopathological tissue damage. Absence of Chlamydia-specific TH2 immunity in these mice newly highlights the need to identify experimental models of C. trachomatis genital infection that more closely recapitulate the human host response., Competing Interests: The authors have declared that no competing interests exist.