Persistent Chlamydia trachomatis Infection of HeLa Cells Mediates Apoptosis Resistance through a Chlamydia Protease-Like Activity Factor-Independent Mechanism and Induces High Mobility Group Box 1 Release

ABSTRACTIntracellular persistence ofChlamydia trachomatishas been implicated in the development of chronic infection that can result in pelvic inflammatory disease and tubal sterility. By inhibition of host cell apoptosis, chlamydiae have evolved a strategy to maintain the intracellular environment for replication and persistence. Both antiapoptotic host cell-derived factors and the chlamydial protease-like activity factor (CPAF) are involved inChlamydia-mediated apoptosis resistance. Here, we show that in HeLa cells infected with gamma interferon (IFN-γ)-induced persistentC. trachomatisserovar D, the expression of CPAF is downregulated, and proapoptotic protease substrates are not cleaved. Persistent infection protected HeLa cells from apoptosis when they were exposed to staurosporine. Small-interfering RNA-mediated inhibition of myeloid cell leukemia 1 (Mcl-1) protein upregulation sensitized persistently infected cells for apoptosis. The inhibitor of apoptosis protein 2 (IAP-2) seems not to be relevant in this context because IAP-2 protein was not induced in response to IFN-γ treatment. Although apoptosis was inhibited, persistent infection caused cell membrane disintegration, as measured by the increased release of cytokeratin 18 from HeLa cells. Moreover, persistently infected cells released significantly increased amounts of high mobility group box 1 (HMGB1) protein which represents a proinflammatory damage-associated pattern molecule. The data of this study suggest that cells infected with persistentC. trachomatisare protected from apoptosis independently of CPAF but may promote chronic inflammation through HMGB1 release.

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Role of High-Mobility Group Box 1 Protein and Poly(ADP-Ribose) Polymerase 1 Degradation in Chlamydia trachomatis-Induced Cytopathicity

Infection and Immunity ◽

10.1128/iai.01404-09 ◽

2010 ◽

Vol 78 (7) ◽

pp. 3288-3297 ◽

Cited By ~ 19

Author(s):

Hangxing Yu ◽

Katja Schwarzer ◽

Martin Förster ◽

Olaf Kniemeyer ◽

Vera Forsbach-Birk ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Cell Membrane ◽

Hela Cells ◽

High Mobility ◽

Host Cells ◽

High Mobility Group ◽

Cell Membrane Permeability ◽

Dependent Manner ◽

Infected Cells ◽

Parp 1

ABSTRACT As intracellular bacteria, chlamydiae block the apoptotic pathways of their host cells. However, the infection of epithelial cells causes the loss of cell membrane integrity and can result in nonapoptotic death. Normally, cells undergoing necrosis release high-mobility group box 1 protein (HMGB1) that acts as an important proinflammatory mediator. Here, we show that in Chlamydia trachomatis-infected HeLa cells HMGB1 is not translocated from the nucleus to the cytosol and not released from injured cells in increased amounts. At 48 h after infection, degradation of HMGB1 was observed. In infected cells, poly(ADP-ribose) polymerase 1 (PARP-1), a DNA repair enzyme that also regulates HMGB1 translocation, was found to be cleaved into fragments that correspond to a necrosislike pattern of PARP-1 degradation. Cell-free cleavage assays and immunoprecipitation using purified proteolytic fractions from infected cells demonstrated that the chlamydial-protease-like activity factor (CPAF) is responsible for the cleavage of both HMGB1 and PARP-1. Proteolytic cleavage of PARP-1 was accompanied by a significant decrease in the enzymatic activity in a time-dependent manner. The loss of PARP-1 function obviously affects the viability of Chlamydia-infected cells because silencing of PARP-1 in uninfected HeLa cells with specific small interfering RNA results in increased cell membrane permeability. Our findings suggest that the Chlamydia-specific protease CPAF interferes with necrotic cell death pathways. By the degradation of HMGB1 and PARP-1, the pathogen may have evolved a strategy to reduce the inflammatory response to membrane-damaged cells in vivo.

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A Chlamydia trachomatis OmcB C-Terminal Fragment Is Released into the Host Cell Cytoplasm and Is Immunogenic in Humans

Infection and Immunity ◽

10.1128/iai.00003-11 ◽

2011 ◽

Vol 79 (6) ◽

pp. 2193-2203 ◽

Cited By ~ 10

Author(s):

Manli Qi ◽

Siqi Gong ◽

Lei Lei ◽

Quanzhong Liu ◽

Guangming Zhong

Keyword(s):

Chlamydia Trachomatis ◽

Host Cell ◽

Host Cells ◽

Content Type ◽

Membrane Complex ◽

Host Cell Cytoplasm ◽

Complex Protein ◽

Infected Cells ◽

Cell Cytosol ◽

Host Cell Cytosol

ABSTRACTTheChlamydia trachomatisouter membrane complex protein B (OmcB) is an antigen with diagnostic and vaccine relevance. To further characterize OmcB, we generated antibodies against OmcB C-terminal (OmcBc) and N-terminal (OmcBn) fragments. Surprisingly, the anti-OmcBc antibody detected dominant signals in the host cell cytosol, while the anti-OmcBn antibody exclusively labeled intrainclusion signals inC. trachomatis-infected cells permeabilized with saponin. Western blot analyses revealed that OmcB was partially processed into OmcBc and OmcBn fragments. The processed OmcBc was released into host cell cytosol, while the OmcBn and remaining full-length OmcB were retained within the chlamydial inclusions. The organism-associated OmcB epitopes became detectable only after theC. trachomatis-infected cells were permeabilized with strong detergents such as SDS. However, the harsh permeabilization conditions also led to the leakage of the already secreted OmcBc and chlamydia-secreted protease (CPAF) out of the host cells. The OmcBc processing and release occurred in all biovars ofC. trachomatis. Moreover, the released OmcBc but not the retained OmcBn was highly immunogenic inC. trachomatis-infected women, which is consistent with the concept that exposure of chlamydial proteins to host cell cytosol is accompanied by increased immunogenicity. These observations have provided important information for further exploring/optimizing OmcB as a target for the development of diagnosis methods and vaccines.

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Infection of HeLa cells with Chlamydia trachomatis inhibits protein synthesis and causes multiple changes to host cell pathways

Cellular Microbiology ◽

10.1111/cmi.12993 ◽

2019 ◽

Vol 21 (4) ◽

Cited By ~ 3

Author(s):

Michaela Ohmer ◽

Tina Tzivelekidis ◽

Nora Niedenführ ◽

Larisa Volceanov‐Hahn ◽

Svenja Barth ◽

...

Keyword(s):

Protein Synthesis ◽

Chlamydia Trachomatis ◽

Host Cell ◽

Hela Cells

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Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion

Infection and Immunity ◽

10.1128/iai.00841-19 ◽

2020 ◽

Vol 88 (5) ◽

Author(s):

Susmita Ghosh ◽

Elizabeth A. Ruelke ◽

Joshua C. Ferrell ◽

Maria D. Bodero ◽

Kenneth A. Fields ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Host Cell ◽

Host Cells ◽

Actin Binding ◽

Wild Type ◽

Content Type ◽

Allelic Exchange ◽

Binding Domains

ABSTRACT The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

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Selected Immunological Mediators and Cervical Microbial Signatures in Women with Chlamydia trachomatis Infection

mSystems ◽

10.1128/msystems.00094-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 5

Author(s):

Simone Filardo ◽

Marisa Di Pietro ◽

Giulia Tranquilli ◽

Maria Agnese Latino ◽

Nadia Recine ◽

...

Keyword(s):

Immune System ◽

Chlamydia Trachomatis ◽

Host Immune System ◽

Complex Interplay ◽

Content Type ◽

Immune Mediators ◽

Potential Biomarker ◽

Ifn Γ ◽

Low Levels ◽

Female Genital

ABSTRACT In the female genital ecosystem, the complex interplay between the host immune system and the resident microflora protects against urogenital pathogens, like Chlamydia trachomatis. C. trachomatis is responsible for urethritis and cervicitis; however, most chlamydial infections are asymptomatic and, thus, not treated, potentially leading to severe reproductive sequelae. Here we investigated the interaction between the levels of selected immune mediators and the community state types of the cervical microbiota in C. trachomatis-infected women. Cervical samples from 42 C. trachomatis-positive women and 103 matched healthy controls were analyzed through the metagenomic analysis of the hypervariable region v4 of the 16S rRNA gene and the determination of lactoferrin, interleukin 1α (IL-1α), IL-6, alpha interferon (IFN-α), IFN-β, and IFN-γ by ELISA. Overall, C. trachomatis infection was significantly associated with a microbiota dominated by anaerobic bacteria (P = 0.000002). In addition, a network of Gardnerella vaginalis, Prevotella amnii, Prevotella buccalis, Prevotella timonensis, Aerococcus christensenii, and Variovorax guangxiensis has been identified as a potential biomarker of C. trachomatis infection through multiple statistical approaches. Again, chlamydial infection was significantly correlated with an increased production of lactoferrin, IL-6, IL-1α, IFN-α, and IFN-β (P < 0.05), whereas very low levels of IFN-γ were observed in C. trachomatis-infected women, levels similar to those detected in healthy women. Our findings show a distinctive signature of C. trachomatis genital infection, characterized by a specific bacterial network, constituted by anaerobes, as well as by increased levels of lactoferrin and proinflammatory cytokines (IL-1α, IL-6, IFN-α, and IFN-β), accompanied by low levels of IFN-γ. IMPORTANCE To our knowledge, this is the first study that investigated the association of C. trachomatis with the cervical levels of lactoferrin and selected inflammatory mediators and their correlation with the different community state types characterizing the female genital ecosystem. C. trachomatis, known as the leading cause of bacterial sexually transmitted diseases, continues to be an important public health problem worldwide for its increasing incidence and the risk of developing severe reproductive sequelae, like pelvic inflammatory disease and infertility. Specifically, C. trachomatis tend to persist in the female genital tract, leading to a chronic inflammatory state characterized by increased production of immune mediators responsible for tissue damage. Therefore, our study may help to broaden the knowledge on the complex interplay between the female genital microbiota and the host immune system in response to C. trachomatis infection.

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Antiapoptotic activity of Chlamydia trachomatis Pgp3 protein involves activation of the ERK1/2 pathway mediated by upregulation of DJ-1 protein

Pathogens and Disease ◽

10.1093/femspd/ftaa003 ◽

2019 ◽

Vol 77 (9) ◽

Cited By ~ 1

Author(s):

Fangzhen Luo ◽

Mingyi Shu ◽

Silu Gong ◽

Yating Wen ◽

Bei He ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Hela Cells ◽

Signal Pathway ◽

Immune Defense ◽

Apoptosis Resistance ◽

Host Cell Apoptosis ◽

Pathway Activation ◽

Tnf Α ◽

Antiapoptotic Activity

ABSTRACT 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.

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Gamma Interferon Is Required for Chlamydia Clearance but Is Dispensable for T Cell Homing to the Genital Tract

mBio ◽

10.1128/mbio.00191-20 ◽

2020 ◽

Vol 11 (2) ◽

Author(s):

Jennifer D. Helble ◽

Rodrigo J. Gonzalez ◽

Ulrich H. von Andrian ◽

Michael N. Starnbach

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Chlamydia Trachomatis ◽

Genital Tract ◽

Gamma Interferon ◽

Cell Homing ◽

Content Type ◽

T Cell Homing ◽

Ifn Γ

ABSTRACT While there is no effective vaccine against Chlamydia trachomatis infection, previous work has demonstrated the importance of C. trachomatis-specific CD4+ T cells (NR1 T cells) in pathogen clearance. Specifically, NR1 T cells have been shown to be protective in mice, and this protection depends on the host’s ability to sense the cytokine gamma interferon (IFN-γ). However, it is unclear what role NR1 production or sensing of IFN-γ plays in T cell homing to the genital tract or T cell-mediated protection against C. trachomatis. Using two-photon microscopy and flow cytometry, we found that naive wild-type (WT), IFN-γ−/−, and IFN-γR−/− NR1 T cells specifically home to sections in the genital tract that contain C. trachomatis. We also determined that protection against infection requires production of IFN-γ from either NR1 T cells or endogenous cells, further highlighting the importance of IFN-γ in clearing C. trachomatis infection. IMPORTANCE Chlamydia trachomatis is an important mucosal pathogen that is the leading cause of sexually transmitted bacterial infections in the United States. Despite this, there is no vaccine currently available. In order to develop such a vaccine, it is necessary to understand the components of the immune response that can lead to protection against this pathogen. It is well known that antigen-specific CD4+ T cells are critical for Chlamydia clearance, but the contexts in which they are protective or not protective are unknown. Here, we aimed to characterize the importance of gamma interferon production and sensing by T cells and the effects on the immune response to C. trachomatis. Our work here helps to define the contexts in which antigen-specific T cells can be protective, which is critical to our ability to design an effective and protective vaccine against C. trachomatis.

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GABARAPL2 Is Critical for Growth Restriction of Toxoplasma gondii in HeLa Cells Treated with Gamma Interferon

Infection and Immunity ◽

10.1128/iai.00054-20 ◽

2020 ◽

Vol 88 (5) ◽

Author(s):

Zhaoxia Zhang ◽

Haorong Gu ◽

Qi Li ◽

Jun Zheng ◽

Shinuo Cao ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Hela Cells ◽

Critical Role ◽

Gamma Interferon ◽

Growth Restriction ◽

Membrane Structures ◽

Content Type ◽

Receptor Associated Protein ◽

Ifn Γ ◽

And Function

ABSTRACT Gamma interferon (IFN-γ)-induced innate immune responses play important roles in the inhibition of Toxoplasma gondii infection. It has been reported that IFN-γ stimulates non-acidification-dependent growth restriction of T. gondii in HeLa cells, but the mechanism remains unclear. Here, we found that γ-aminobutyric acid (GABA) receptor-associated protein-like 2 (GABARAPL2) plays a critical role in parasite restriction in IFN-γ-treated HeLa cells. GABARAPL2 is recruited to membrane structures surrounding parasitophorous vacuoles (PV). Autophagy adaptors are required for the proper localization and function of GABARAPL2 in the IFN-γ -induced immune response. These findings provide further understanding of a noncanonical autophagy pathway responsible for IFN-γ-dependent inhibition of T. gondii growth in human HeLa cells and demonstrate the critical role of GABARAPL2 in this response.

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Inhibition of tRNA Synthetases Induces Persistence in Chlamydia

Infection and Immunity ◽

10.1128/iai.00943-19 ◽

2020 ◽

Vol 88 (4) ◽

Cited By ~ 1

Author(s):

Nathan D. Hatch ◽

Scot P. Ouellette

Keyword(s):

Host Cell ◽

Chlamydia Pneumoniae ◽

Respiratory Infections ◽

Developmental State ◽

Trna Synthetase ◽

Host Cells ◽

Host Immune System ◽

Content Type ◽

Ifn Γ

ABSTRACT Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections, and Chlamydia pneumoniae causes community-acquired respiratory infections. In vivo, the host immune system will release gamma interferon (IFN-γ) to combat infection. IFN-γ activates human cells to produce the tryptophan (Trp)-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). Consequently, there is a reduction in cytosolic Trp in IFN-γ-activated host cells. In evolving to obligate intracellular dependence, Chlamydia has significantly reduced its genome size and content, as it relies on the host cell for various nutrients. Importantly, C. trachomatis and C. pneumoniae are Trp auxotrophs and are starved for this essential nutrient when the human host cell is exposed to IFN-γ. To survive this, chlamydiae enter an alternative developmental state referred to as persistence. Chlamydial persistence is characterized by a halt in the division cycle, aberrant morphology, and, in the case of IFN-γ-induced persistence, Trp codon-dependent changes in transcription. We hypothesize that these changes in transcription are dependent on the particular amino acid starvation state. To investigate the chlamydial response mechanisms acting when other amino acids become limiting, we tested the efficacy of prokaryote-specific tRNA synthetase inhibitors, indolmycin and AN3365, to mimic starvation of Trp and leucine, respectively. We show that these drugs block chlamydial growth and induce changes in morphology and transcription consistent with persistence. Importantly, growth inhibition was reversed when the compounds were removed from the medium. With these data, we find that indolmycin and AN3365 are valid tools that can be used to mimic the persistent state independently of IFN-γ.

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Inhibition of Apoptosis by Gamma Interferon in Cells and Mice Infected with Chlamydia muridarum (the Mouse Pneumonitis Strain of Chlamydia trachomatis)

Infection and Immunity ◽

10.1128/iai.70.5.2559-2565.2002 ◽

2002 ◽

Vol 70 (5) ◽

pp. 2559-2565 ◽

Cited By ~ 14

Author(s):

Jean-Luc Perfettini ◽

Toni Darville ◽

Alice Dautry-Varsat ◽

Roger G. Rank ◽

David M. Ojcius

Keyword(s):

Chlamydia Trachomatis ◽

Inhibitory Effect ◽

Gamma Interferon ◽

Apoptotic Cells ◽

Chlamydia Muridarum ◽

Infected Cells ◽

Heterogenous Population ◽

Ifn Γ ◽

In Cells

ABSTRACT The effect of gamma interferon (IFN-γ) on apoptosis due to infection by Chlamydia muridarum (the mouse pneumonitis strain of Chlamydia trachomatis) was studied in epithelial cells in culture and in the genital tracts of mice. IFN-γ concentrations that induce the formation of aberrant, persistent chlamydiae inhibit apoptosis due to C. muridarum infection. In cells treated with an IFN-γ concentration that leads to the development of a heterogenous population of normal and aberrant Chlamydia vacuoles, apoptosis was inhibited preferentially in cells that contained the aberrant vacuoles. The inhibitory effect of IFN-γ appears to be due in part to expression of host cell indoleamine 2,3-dioxygenase activity, since inhibition of apoptosis could be partially reversed through coincubation with exogenous tryptophan. Apoptotic cells were observed in the genital tracts of wild-type mice infected with C. muridarum, and a significantly larger number of apoptotic cells was detected in infected IFN-γ-deficient mice. These results suggest that IFN-γ may contribute to pathogenesis of persistent Chlamydia infections in vivo by preventing apoptosis of infected cells.

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