Natural Clearance of Chlamydia trachomatis Infection Is Associated With Distinct Differences in Cervicovaginal Metabolites.

BACKGROUND: Natural clearance of Chlamydia trachomatis in women occurs in the interval between screening and treatment. In vitro, interferon-γ (IFN-γ)-mediated tryptophan depletion results in C. trachomatis clearance, but whether this mechanism occurs in vivo remains unclear. We previously found that women who naturally cleared C. trachomatis had lower cervicovaginal levels of tryptophan and IFN-γ compared to women with persisting infection, suggesting IFN-γ-independent pathways may promote C. trachomatis clearance. METHODS: Cervicovaginal lavages from 34 women who did (n = 17) or did not (n = 17) naturally clear C. trachomatis were subjected to untargeted high-performance liquid chromatography mass-spectrometry to identify metabolites and metabolic pathways associated with natural clearance. RESULTS: In total, 375 positively charged metabolites and 149 negatively charged metabolites were annotated. Compared to women with persisting infection, C. trachomatis natural clearance was associated with increased levels of oligosaccharides trehalose, sucrose, melezitose, and maltotriose, and lower levels of indoline and various amino acids. Metabolites were associated with valine, leucine, and isoleucine biosynthesis pathways. CONCLUSIONS: The cervicovaginal metabolome in women who did or did not naturally clear C. trachomatis is distinct. In women who cleared C. trachomatis, depletion of various amino acids, especially valine, leucine, and isoleucine, suggests that amino acids other than tryptophan impact C. trachomatis survival in vivo.

Chlamydia trachomatis induces the transcriptional activity of host YAP in a Hippo-independent fashion.

Introduction: The obligate intracellular pathogen Chlamydia trachomatis is the causative agent of the most common bacterial sexually transmitted disease worldwide. While the host response to infection by this pathogen has been well characterized, it remains unclear to what extent host gene expression during infection is the product of Chlamydia-directed modulation of host transcription factors. Methods: To identify transcription factors potentially modulated by Chlamydia during infection, we infected immortalized endocervical epithelial cells (End1/E6E7) with the anogenital C. trachomatis serovar L2, harvesting polyadenylated RNA for bulk RNA-sequencing. Subsequent experiments elucidating the mechanism of infection-mediated YAP activation assayed YAP target gene expression via qRT-PCR, YAP nuclear translocation via quantitative immunofluorescence, and YAP phosphorylation via Western blotting. Results: RNA sequencing of Chlamydia-infected endocervical epithelial cells revealed gene expression consistent with activity of YAP, a transcriptional coactivator implicated in cell proliferation, wound healing, and fibrosis. After confirming induction of YAP target genes during infection, we observed an infection-dependent increase in YAP nuclear translocation sensitive to inhibition of bacterial protein synthesis. While Hippo-mediated phosphoinhibition of YAP at S127 was unaffected by C. trachomatis infection, Hippo-independent phosphorylation at Y357 was increased. Infection did not enhance nuclear translocation of Y357F mutant YAP, illustrating a requirement for phosphorylation at this residue. Pharmacological inhibition of host Src-family kinase activity attenuated YAP Y357 phosphorylation, but not nuclear translocation - which was instead sensitive to inhibition of Abl. Discussion: Our results define a transcriptome-altering mechanism of pathogen-directed YAP activation that bypasses canonical inhibition by the Hippo kinase cascade, with a potential link to chlamydial fibrosis and other advanced disease sequelae. Additional study is required to determine the specific role of infection-associated Y357 phosphorylation and Abl activity in chlamydial induction of YAP.

Chlamydia repurposes the actin-binding protein EPS8 to disassemble epithelial tight junctions and promote infection.

Invasive microbial pathogens often disrupt epithelial barriers, yet the mechanisms used to dismantle tight junctions are poorly understood. Here, we show that the obligate pathogen Chlamydia trachomatis uses the effector protein TepP to transiently disassemble tight junctions early during infection. TepP alters the tyrosine phosphorylation status of host proteins involved in cytoskeletal regulation, including the filamentous actin-binding protein EPS8. We determined that TepP and EPS8 are necessary and sufficient to remodel tight junctions and that the ensuing disruption of epithelial barrier function promotes secondary invasion events. The genetic deletion of EPS8 renders epithelial cells and endometrial organoids resistant to TepP-mediated tight junction remodeling. Finally, TepP and EPS8 promote infection in murine models of infections, with TepP mutants displaying defects in ascension to the upper genital tract. These findings reveal a non-canonical function of EPS8 in the disassembly of epithelial junctions and an important role for Chlamydia pathogenesis.

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.

Chlamydia trachomatis development requires both host glycolysis and oxidative phosphorylation but has only minor effects on these pathways.

The obligate intracellular bacteria Chlamydia trachomatis obtain all nutrients from the cytoplasm of their epithelial host cells and stimulate glucose uptake by these cells. They even hijack host ATP, exerting a strong metabolic pressure on their host at the peak of the proliferative stage of their developmental cycle. However, it is largely unknown whether infection modulates the metabolism of the host cell. Also, the reliance of the bacteria on host metabolism might change during their progression through their biphasic developmental cycle. Herein, using primary epithelial cells and 2 cell lines of nontumoral origin, we showed that between the 2 main ATP-producing pathways of the host, oxidative phosphorylation (OxPhos) remained stable and glycolysis was slightly increased. Inhibition of either pathway strongly reduced bacterial proliferation, implicating that optimal bacterial growth required both pathways to function at full capacity. While we found C. trachomatis displayed some degree of energetic autonomy in the synthesis of proteins expressed at the onset of infection, functional host glycolysis was necessary for the establishment of early inclusions, whereas OxPhos contributed less. These observations correlated with the relative contributions of the pathways in maintaining ATP levels in epithelial cells, with glycolysis contributing the most. Altogether, this work highlights the dependence of C. trachomatis on both host glycolysis and OxPhos for efficient bacterial replication. However, ATP consumption appears at equilibrium with the normal production capacity of the host and the bacteria, so that no major shift between these pathways is required to meet bacterial needs.

Characterization of Pathogenic CD8+ T cells in Chlamydia-Infected OT1 Mice.

Chlamydia trachomatis is a leading infectious cause of infertility in women due to its induction of lasting pathology such as hydrosalpinx. Chlamydia muridarum induces mouse hydrosalpinx because C. muridarum can both invade tubal epithelia directly (as a first hit) and induce lymphocytes to promote hydrosalpinx indirectly (as a second hit). In the current study, a critical role of CD8+ T cells in chlamydial induction of hydrosalpinx was validated in both wild type C57BL/6J mice and OT1 transgenic mice. OT1 mice failed to develop hydrosalpinx partially due to the failure of their lymphocytes to recognize chlamydial antigens. CD8+ T cells from naive C57BL/6J mice rescued the ability of recipient OT1 mice to develop hydrosalpinx when naive CD8+ T cells were transferred at the time of infection with Chlamydia. However, when the transfer was delayed for 2 weeks or longer after the Chlamydia infection, naive CD8+ T cells no longer promoted hydrosalpinx. Nevertheless, CD8+ T cells from mice immunized against Chlamydia still promoted significant hydrosalpinx in the recipient OT1 mice even when the transfer was delayed for 3 weeks. Thus, CD8+ T cells must be primed within 2 weeks after Chlamydia infection to be pathogenic, but, once primed, they can promote hydrosalpinx for >3 weeks. However, Chlamydia-primed CD4+ T cells failed to promote chlamydial induction of pathology in OT1 mice. This study optimized an OT1 mouse-based model for revealing the pathogenic mechanisms of Chlamydia-specific CD8+ T cells.

Cross Talk between ARF1 and RhoA Coordinates the Formation of Cytoskeletal Scaffolds during Chlamydia Infection.

Chlamydia trachomatis is an obligate intracellular bacterium that has developed sophisticated mechanisms to survive inside its infectious compartment, the inclusion. Notably, Chlamydia weaves an extensive network of microtubules (MTs) and actin filaments to enable interactions with host organelles and enhance its stability. Despite the global health and economic burden caused by this sexually transmitted pathogen, little is known about how actin and MT scaffolds are integrated into an increasingly complex virulence system. Previously, we established that the chlamydial effector InaC interacts with ARF1 to stabilize MTs. We now demonstrate that InaC regulates RhoA to control actin scaffolds. InaC relies on cross talk between ARF1 and RhoA to coordinate MTs and actin, where the presence of RhoA downregulates stable MT scaffolds and ARF1 activation inhibits actin scaffolds. Understanding how Chlamydia hijacks complex networks will help elucidate how this clinically significant pathogen parasitizes its host and reveal novel cellular signaling pathways. IMPORTANCE Chlamydia trachomatis is a major cause of human disease worldwide. The ability of Chlamydia to establish infection and cause disease depends on the maintenance of its parasitic niche, called the inclusion. To accomplish this feat, Chlamydia reorganizes host actin and microtubules around the inclusion membrane. How Chlamydia orchestrates these complex processes, however, is largely unknown. Here, we discovered that the chlamydial effector InaC activates Ras homolog family member A (RhoA) to control the formation of actin scaffolds around the inclusion, an event that is critical for inclusion stability. Furthermore, InaC directs the kinetics of actin and posttranslationally modified microtubule scaffolds by mediating cross talk between the GTPases that control these cytoskeletal elements, RhoA and ADP-ribosylation factor 1 (ARF1). The precise timing of these events is essential for the maintenance of the inclusion. Overall, this study provides the first evidence of ARF1-RhoA-mediated cross talk by a bacterial pathogen to coopt the host cytoskeleton.

Chlamydia evasion of neutrophil host defense results in NLRP3 dependent myeloid-mediated sterile inflammation through the purinergic P2X7 receptor.

Chlamydia trachomatis infection causes severe inflammatory disease resulting in blindness and infertility. The pathophysiology of these diseases remains elusive but myeloid cell-associated inflammation has been implicated. Here we show NLRP3 inflammasome activation is essential for driving a macrophage-associated endometritis resulting in infertility by using a female mouse genital tract chlamydial infection model. We find the chlamydial parasitophorous vacuole protein CT135 triggers NLRP3 inflammasome activation via TLR2/MyD88 signaling as a pathogenic strategy to evade neutrophil host defense. Paradoxically, a consequence of CT135 mediated neutrophil killing results in a submucosal macrophage-associated endometritis driven by ATP/P2X7R induced NLRP3 inflammasome activation. Importantly, macrophage-associated immunopathology occurs independent of macrophage infection. We show chlamydial infection of neutrophils and epithelial cells produce elevated levels of extracellular ATP. We propose this source of ATP serves as a DAMP to activate submucosal macrophage NLRP3 inflammasome that drive damaging immunopathology. These findings offer a paradigm of sterile inflammation in infectious disease pathogenesis.

Tumor Necrosis Factor Alpha-Induced Interleukin-1 Alpha Synthesis and Cell Death Is Increased in Mouse Epithelial Cells Infected With Chlamydia muridarum.

Chlamydia trachomatis-genital infection in women can be modeled in mice using Chlamydia muridarum. Using this model, it has been shown that the cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1α lead to irreversible tissue damage in the oviducts. In this study, we investigated the contribution of TNFα on IL-1α synthesis in infected epithelial cells. We show that C muridarum infection enhanced TNFα-induced IL-1α expression and release in a mouse epithelial cell line. In addition to IL-1α, several TNFα-induced inflammatory genes were also highly induced, and infection enhanced TNF-induced cell death. In the mouse model of genital infection, oviducts from mice lacking the TNFα receptor displayed minimal staining for IL-1α compared with wild-type oviducts. Our results suggest TNFα and IL-1α enhance each other's downstream effects resulting in a hyperinflammatory response to chlamydial infection. We propose that biologics targeting TNF-induced IL-1α synthesis could be used to mitigate tissue damage during chlamydial infection.

Role of Interleukin-4 (IL-4) in Respiratory Infection and Allergy Caused by Early-Life Chlamydia Infection.

Chlamydia pneumoniae is a type of pathogenic gram-negative bacteria that causes various respiratory tract infections including asthma. Chlamydia species infect humans and cause respiratory infection by rupturing the lining of the respiratory which includes the throat, lungs and windpipe. Meanwhile, the function of interleukin-4 (IL-4) in Ch. pneumoniae respiratory infection and its association with the development of airway hyperresponsiveness (AHR) in adulthood and causing allergic airway disease (AAD) are not understood properly. We therefore investigated the role of IL-4 in respiratory infection and allergy caused by early life Chlamydia infection. In this study, Ch. pneumonia strain was propagated and cultured in HEp-2 cells according to standard protocol and infant C57BL/6 mice around 3-4 weeks old were infected to study the role of IL-4 in respiratory infection and allergy caused by early life Chlamydia infection. We observed that IL-4 is linked with Chlamydia respiratory infection and its absence lowers respiratory infection. IL-4R α2 is also responsible for controlling the IL-4 signaling pathway and averts the progression of infection and inflammation. Furthermore, the IL-4 signaling pathway also influences infection-induced AHR and aids in increasing AAD severity. STAT6 also promotes respiratory infection caused by Ch. pneumoniae and further enhanced its downstream process. Our study concluded that IL-4 is a potential target for preventing infection-induced AHR and severe asthma.

Shifting proteomes: limitations in using the BioID proximity labeling system to study SNARE protein trafficking during infection with intracellular pathogens.

We hypothesize that intracellular trafficking pathways are altered in chlamydial infected cells to maximize the ability of Chlamydia to scavenge nutrients while not overtly stressing the host cell. Previous data demonstrated the importance of two eukaryotic SNARE proteins, VAMP4 and syntaxin 10 (Stx10), in chlamydial growth and development. Although, the mechanism for these effects is still unknown. To interrogate whether chlamydial infection altered these proteins' networks, we created BirA*-VAMP4 and BirA*-Stx10 fusion constructs to use the BioID proximity labeling system. While we identified a novel eukaryotic protein-protein interaction between Stx10 and VAPB, we also identified caveats in using the BioID system to study the impact of infection by an obligate intracellular pathogen on SNARE protein networks. The addition of the BirA* altered the localization of VAMP4 and Stx10 during infection with Chlamydia trachomatis serovars L2 and D and Coxiella burnetii Nine Mile Phase II. We also discovered that BirA* traffics to and biotinylates Coxiella-containing vacuoles and, in general, has a propensity for labeling membrane or membrane-associated proteins. While the BioID system identified a novel association for Stx10, it is not a reliable methodology to examine intracellular trafficking pathway dynamics during infection with intracellular pathogens.

Reduced Uterine Tissue Damage during Chlamydia muridarum Infection in TREM-1,3-Deficient Mice.

Genital infections with Chlamydia trachomatis can lead to uterine and oviduct tissue damage in the female reproductive tract. Neutrophils are strongly associated with tissue damage during chlamydial infection, while an adaptive CD4 T cell response is necessary to combat infection. Activation of triggering receptor expressed on myeloid cells-1 (TREM-1) on neutrophils has previously been shown to induce and/or enhance degranulation synergistically with Toll-like receptor (TLR) signaling. Additionally, TREM-1 can promote neutrophil transepithelial migration. In this study, we sought to determine the contribution of TREM-1,3 to immunopathology in the female mouse genital tract during Chlamydia muridarum infection. Relative to control mice, trem1,3-/- mice had no difference in chlamydial burden or duration of lower-genital-tract infection. We also observed a similar incidence of hydrosalpinx 45 days postinfection in trem1,3-/- compared to wild-type (WT) mice. However, compared to WT mice, trem1,3-/- mice developed significantly fewer hydrometra in uterine horns. Early in infection, trem1,3-/- mice displayed a notable decrease in the number of uterine glands containing polymorphonuclear cells and uterine horn lumens had fewer neutrophils, with increased granulocyte colony-stimulating factor (G-CSF). trem1,3-/- mice also had reduced erosion of the luminal epithelium. These data indicate that TREM-1,3 contributes to transepithelial neutrophil migration in the uterus and uterine glands, promoting the occurrence of hydrometra in infected mice.

Akt Phosphorylation Influences Persistent Chlamydial Infection and Chlamydia-Induced Golgi Fragmentation Without Involving Rab14.

Chlamydia trachomatis is an obligate intracellular bacterium that causes multiple diseases involving the eyes, gastrointestinal tract, and genitourinary system. Previous studies have identified that in acute chlamydial infection, C. trachomatis requires Akt pathway phosphorylation and Rab14-positive vesicles to transmit essential lipids from the Golgi apparatus in survival and replication. However, the roles that Akt phosphorylation and Rab14 play in persistent chlamydial infection remain unclear. Here, we discovered that the level of Akt phosphorylation was lower in persistent chlamydial infection, and positively correlated with the effect of activating the development of Chlamydia but did not change the infectivity and 16s rRNA gene expression. Rab14 was found to exert a limited effect on persistent infection. Akt phosphorylation might regulate Chlamydia development and Chlamydia-induced Golgi fragmentation in persistent infection without involving Rab14. Our results provide a new insight regarding the potential of synergistic repressive effects of an Akt inhibitor with antibiotics in the treatment of persistent chlamydial infection induced by penicillin.

Complement and Chlamydia psittaci: Non-Myeloid-Derived C3 Predominantly Induces Protective Adaptive Immune Responses in Mouse Lung Infection.

Recent advances in complement research have revolutionized our understanding of its role in immune responses. The immunomodulatory features of complement in infections by intracellular pathogens, e.g., viruses, are attracting increasing attention. Thereby, local production and activation of complement by myeloid-derived cells seem to be crucial. We could recently show that C3, a key player of the complement cascade, is required for effective defense against the intracellular bacterium Chlamydia psittaci. Avian zoonotic strains of this pathogen cause life-threatening pneumonia with systemic spread in humans; closely related non-avian strains are responsible for less severe diseases of domestic animals with economic loss. To clarify how far myeloid- and non-myeloid cell-derived complement contributes to immune response and resulting protection against C. psittaci, adoptive bone marrow transfer experiments focusing on C3 were combined with challenge experiments using a non-avian (BSL 2) strain of this intracellular bacterium. Surprisingly, our data prove that for C. psittaci-induced pneumonia in mice, non-myeloid-derived, circulating/systemic C3 has a leading role in protection, in particular on the development of pathogen-specific T- and B- cell responses. In contrast, myeloid-derived and most likely locally produced C3 plays only a minor, mainly fine-tuning role. The work we present here describes authentic, although less pronounced, antigen directed immune responses.

SND1 promotes Th1/17 immunity against chlamydial lung infection through enhancing dendritic cell function.

To date, no reports have linked the multifunctional protein, staphylococcal nuclease domain-containing protein 1 (SND1), to host defense against intracellular infections. In this study, we investigated the role and mechanisms of SND1, by using SND1 knockout (SND1-/-) mice, in host defense against the lung infection of Chlamydia muridarum, an obligate intracellular bacterium. Our data showed that SND1-/- mice exhibited significantly greater body weight loss, higher organism growth, and more severe pathological changes compared with wild-type mice following the infection. Further analysis showed significantly reduced Chlamydia-specific Th1/17 immune responses in SND1-/- mice after infection. Interestingly, the dendritic cells (DCs) isolated from SND1-/- mice showed lower costimulatory molecules expression and IL-12 production, but higher IL-10 production compared with those from wild-type control mice. In the DC-T cell co-culture system, DCs isolated from SND1-/- infected mice showed significantly reduced ability to promote Chlamydia-specific IFN-γ producing Th1 cells but enhanced capacity to induce CD4+T cells into Foxp3+ Treg cells. Adoptive transfer of DCs isolated from SND1-/- mice, unlike those from wild-type control mice, failed to protect the recipients against challenge infection. These findings provide in vivo evidence that SND1 plays an important role in host defense against intracellular bacterial infection, and suggest that SND1 can promote Th1/17 immunity and inhibit the expansion of Treg cells through modulation of the function of DCs.

Active Hexose-Correlated Compound Restores Gene Expression and Protein Secretion of Protective Cytokines of Immune Cells in a Murine Stress Model during Chlamydia muridarum Genital Infection.

Chlamydia trachomatis genital infection is the most common bacterial sexually transmitted disease worldwide. Previously, we reported that cold-induced stress results in immune suppression of mice that subsequently leads to increased intensity of Chlamydia muridarum genital infection. Furthermore, we demonstrated that stressed mice orally fed with active hexose-correlated compound (AHCC) have reduced shedding of C. muridarum from the genital tract. However, the mechanism of AHCC in reducing the organ load and changing the immune response in the stress model is not well known. This study evaluated infection and changes in immunological parameters of stressed AHCC-fed mice with or without C. muridarum genital infection. We hypothesized that AHCC feeding to stressed mice restores protective immune function and reduces susceptibility to C. muridarum genital infection. The results show that oral feeding of stressed mice with AHCC resulted in decreased shedding of C. muridarum from the genital tract, reduced production of plasma catecholamines, increased expression of T-bet and reduced GATA-3 in CD4+ T cells, increased production of interleukin-12 (IL-12) and interferon gamma (IFN-γ) and reduced production of IL-4 in CD4+ T cells, and enhanced expression of surface markers and costimulatory molecules of CD4+ T cells, bone marrow-derived dendritic cells (BMDCs), and natural killer cells. Coculturing of mature BMDCs with splenic CD4+ T cells led to the increased and decreased production of T helper 1 and T helper 2 cytokines, respectively. Overall, our results show that AHCC fosters the restoration of Th1 cytokine production while reducing Th2 cytokine production, which would promote C. muridarum clearance in the murine stress model.

Human ß-defensin-2 suppresses key features of asthma in murine models of allergic airways disease.

BACKGROUND: Asthma is an airway inflammatory disease and a major health problem worldwide. Anti-inflammatory steroids and bronchodilators are the gold-standard therapy for asthma. However, they do not prevent the development of the disease, and critically, a subset of asthmatics are resistant to steroid therapy. OBJECTIVE: To elucidate the therapeutic potential of human ß-defensins (hBD), such as hBD2 mild to moderate and severe asthma. METHODS: We investigated the role of hBD2 in a steroid-sensitive, house dust mite-induced allergic airways disease (AAD) model and a steroid-insensitive model combining ovalbumin-induced AAD with C muridarum (Cmu) respiratory infection. RESULTS: In both models, we demonstrated that therapeutic intranasal application of hBD2 significantly reduced the influx of inflammatory cells into the bronchoalveolar lavage fluid. Furthermore, key type 2 asthma-related cytokines IL-9 and IL-13, as well as additional immunomodulating cytokines, were significantly decreased after administration of hBD2 in the steroid-sensitive model. The suppression of inflammation was associated with improvements in airway physiology and treatment also suppressed airway hyper-responsiveness (AHR) in terms of airway resistance and compliance to methacholine challenge. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that hBD2 reduces the hallmark features and has potential as a new therapeutic agent in allergic and especially steroid-resistant asthma.

Eukaryotic SNARE VAMP3 Dynamically Interacts with Multiple Chlamydial Inclusion Membrane Proteins.

Chlamydia trachomatis, an obligate intracellular pathogen, undergoes a biphasic developmental cycle within a membrane-bound vacuole called the chlamydial inclusion. To facilitate interactions with the host cell, Chlamydia modifies the inclusion membrane with type III secreted proteins, called Incs. As with all chlamydial proteins, Incs are temporally expressed, modifying the chlamydial inclusion during the early and mid-developmental cycle. VAMP3 and VAMP4 are eukaryotic SNARE proteins that mediate membrane fusion and are recruited to the inclusion to facilitate inclusion expansion. Their recruitment requires de novo chlamydial protein synthesis during the mid-developmental cycle. Thus, we hypothesize that VAMP3 and VAMP4 are recruited by Incs. In chlamydia-infected cells, identifying Inc binding partners for SNARE proteins specifically has been elusive. To date, most studies examining chlamydial Inc and eukaryotic proteins have benefitted from stable interacting partners or a robust interaction at a specific time postinfection. While these types of interactions are the predominant class that have been identified, they are likely the exception to chlamydia-host interactions. Therefore, we applied two separate but complementary experimental systems to identify candidate chlamydial Inc binding partners for VAMPs. Based on these results, we created transformed strains of C. trachomatis serovar L2 to inducibly express a candidate Inc-FLAG protein. In chlamydia-infected cells, we found that five Incs temporally and transiently interact with VAMP3. Further, loss of incA or ct813 expression altered VAMP3 localization to the inclusion. For the first time, our studies demonstrate the transient nature of certain host protein-Inc interactions that contribute to the chlamydial developmental cycle.

Inhibition of Chlamydial Infection by CRISPR/Cas9-SAM Mediated Enhancement of Human Peptidoglycan Recognition Proteins Gene Expression in HeLa Cells.

The global problem of emerging resistance of microorganisms to antibiotics makes the search for new natural substances with antibacterial properties relevant. Such substances include peptidoglycan recognition proteins (PGLYRP), which are the components of the innate immunity of many organisms, including humans. These proteins have a unique mechanism of action that allows them to evade the resistance of bacteria to them, as well as to be active against both Gram-positive and Gram-negative bacteria. However, the use of antimicrobial recombinant proteins is not always advisable due to the complexity of local delivery of the proteins and their stability; in this regard it seems appropriate to activate the components of the innate immunity. The aim of this study was to increase the expression level of native peptidoglycan recognition protein genes in HeLa cells using genome-editing technology with synergistic activation mediators (CRISPR/Cas9-SAM) and evaluate antichlamydial effect of PGLYRP. We demonstrated activation of the chlamydial two-component gene system (ctcB-ctcC), which played a key role in the mechanism of action of the peptidoglycan recognition proteins. We generated the HeLa cell line transduced with lentiviruses encoding CRISPR/Cas9-SAM activation system with increased PGLYRP gene expression. It was shown that activation of the own peptidoglycan recognition proteins gene expression in the cell line caused inhibition of the chlamydial infection development. The proposed approach makes it possible to use the capabilities of innate immunity to combat infectious diseases caused by Gram-positive and Gram-negative bacteria.

IL-1α Is Essential for Oviduct Pathology during Genital Chlamydial Infection in Mice.

Chlamydia trachomatis infection of the female genital tract can lead to irreversible fallopian tube scarring. In the mouse model of genital infection using Chlamydia muridarum, IL-1R signaling plays a critical role in oviduct tissue damage. In this study, we investigated the pathologic role of IL-1α, one of the two proinflammatory cytokines that bind to IL-1R. Il1a-/- mice infected with C. muridarum cleared infection at their cervix at the same rate as wild-type (WT) mice, but were significantly protected from end point oviduct damage and fibrosis. The contribution of IL-1α to oviduct pathology was more dramatic than observed in mice deficient for IL-1ß. Although chlamydial burden was similar in WT and Il1a-/- oviduct during peak days of infection, levels of IL-1ß, IL-6, CSF3, and CXCL2 were reduced in Il1a-/- oviduct lysates. During infection, Il1a-/- oviducts and uterine horns exhibited reduced neutrophil infiltration, and this reduction persisted after the infection resolved. The absence of IL-1α did not compromise CD4 T cell recruitment or function during primary or secondary chlamydial infection. IL-1α is expressed predominantly by luminal cells of the genital tract in response to infection, and low levels of expression persisted after the infection cleared. Ab-mediated depletion of IL-1α in WT mice prevented infection-induced oviduct damage, further supporting a key role for IL-1α in oviduct pathology.