Trachomatis Replication Research Articles

Yinjia pill inhibits persistent Chlamydia trachomatis infection.

Yinjia pill inhibits persistent Chlamydia trachomatis infection.

Interferon-γ Possesses Anti-Microbial and Immunomodulatory Activity on a Chlamydia Trachomatis Infection Model of Primary Human Synovial Fibroblasts.

Chlamydia trachomatis, an obligate intracellular pathogen, is the most common cause of bacterial sexually transmitted diseases, and it is potentially responsible for severe chronic sequelae, such as reactive arthritis. To date, details of the mechanisms by which Chlamydiae induce innate antimicrobial pathways in synovial fibroblasts, are not well characterized; therefore, herein, we investigated the effects of interferon (IFN)α, IFNβ, and IFNγ on the infection, and replication phases of the C. trachomatis developmental cycle, as well as on the induction of pattern recognition receptors (PRRs) and IFN-related pathways. To do so, we set up an in vitro chlamydial-infection model of primary human synovial cells treated with IFNs before or after the infection. We then determined the number of chlamydial inclusion forming units and inclusion size, as well as the expression of toll like receptor (TLR)2, TLR3, TLR4, cyclic GMP-AMP synthase (cGAS), stimulator of IFN gene (STING), IRF9, ISG56, and GBP1. The main result of our study is the significant inhibition of C. trachomatis infection and replication in human synovial cells following the treatment with IFNγ, whereas IFN-I proved to be ineffective. Furthermore, IFNγ greatly upregulated all the PRRs and ISGs examined. In conclusion, IFNγ exhibited a potent anti-Chlamydia activity in human synovial cells as well as the ability to induce a strong increase of innate immune pathways.

Growth characteristics of Chlamydia trachomatis in human intestinal epithelial Caco-2 cells.

Chlamydia trachomatis is an obligate intracellular bacterium causing infections of the eyes, urogenital and respiratory tracts. Asymptomatic, repeat and chronic infections with C. trachomatis are common in the urogenital tract potentially causing severe reproductive pathology. Animal models of infection and epidemiological studies suggested the gastrointestinal tract as a reservoir of chlamydiae and as a source of repeat urogenital infections. Thus, we investigated the growth characteristics of C. trachomatis in human intestinal epithelial Caco-2 cells and the infection-induced defensin production. Immunofluorescence staining and transmission electron microscopy showed the presence of chlamydial inclusions in the cells. Chlamydial DNA and viable C. trachomatis were recovered from Caco-2 cells in similar quantity compared to that detected in the usual in vitro host cell of this bacterium. The kinetics of expression of selected C. trachomatis genes in Caco-2 cells indicated prolonged replication with persisting high expression level of late genes and of heat shock protein gene groEL. Replication of C. trachomatis induced moderate level of β-defensin-2 production by Caco-2 cells, which might contribute to avoidance of immune recognition in the intestine. According to our results, Caco-2 cells support C. trachomatis replication, suggesting that the gastrointestinal tract is a site of residence for these bacteria.

Detection of a microbial metabolite by STING regulates inflammasome activation in response to Chlamydia trachomatis infection.

The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and non-infectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1β processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1β processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand—cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted.

The High-Risk Human Papillomavirus E6 Oncogene Exacerbates the Negative Effect of Tryptophan Starvation on the Development of Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular pathogen that requires specific essential nutrients from the host cell, one of which is the amino acid tryptophan. In this context interferon gamma (IFNγ) is the major host protective cytokine against chlamydial infections because it induces the expression of the host enzyme, indoleamine 2,3-dioxygenase 1, that degrades tryptophan, thereby restricting bacterial replication. The mechanism by which IFNγ acts has been dissected in vitro using epithelial cell-lines such as HeLa, HEp-2, or the primary-like endocervical cell-line A2EN. All these cell-lines express the high-risk human papillomavirus oncogenes E6 & E7. While screening cell-lines to identify those suitable for C. trachomatis co-infections with other genital pathogens, we unexpectedly found that tryptophan starvation did not completely block chlamydial development in cell-lines that were HR-HPV negative, such as C33A and 293. Therefore, we tested the hypothesis that HR-HPV oncogenes modulate the effect of tryptophan starvation on chlamydial development by comparing chlamydial development in HeLa and C33A cell-lines that were both derived from cervical carcinomas. Our results indicate that during tryptophan depletion, unlike HeLa, C33A cells generate sufficient intracellular tryptophan via proteasomal activity to permit C. trachomatis replication. By generating stable derivatives of C33A that expressed HPV16 E6, E7 or E6 & E7, we found that E6 expression alone was sufficient to convert C33A cells to behave like HeLa during tryptophan starvation. The reduced tryptophan levels in HeLa cells have a biological consequence; akin to the previously described effect of IFNγ, tryptophan starvation protects C. trachomatis from clearance by doxycycline in HeLa but not C33A cells. Curiously, when compared to the known Homo sapiens proteome, the representation of tryptophan in the HR-HPV E6 & E6AP degradome is substantially lower, possibly providing a mechanism that underlies the lowered intracellular free tryptophan levels in E6-expressing cells during starvation.

Expansion of the Chlamydia trachomatis inclusion does not require bacterial replication

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

Improved plaque assay identifies a novel anti-Chlamydia ceramide derivative with altered intracellular localization.

Chlamydia trachomatis is a medically important human pathogen causing different diseases, including trachoma, the leading cause of preventable blindness in developing countries, and sexually transmitted infections that can lead to infertility and ectopic pregnancies. There is no vaccine against C. trachomatis at present. Broad-spectrum antibiotics are used as standard therapy to treat the infection but have unwanted side effects, such as inducing persistent or recurring infections and affecting the host microbiome, necessitating the development of novel anti-Chlamydia therapies. Here, we describe the establishment of a robust, fast, and simple plaque assay using liquid overlay medium (LOM) for the identification of anti-Chlamydia compounds. Using the LOM plaque assay, we identified nitrobenzoxadiazole (NBD)-labeled 1-O-methyl-ceramide-C16 as a compound that efficiently inhibits C. trachomatis replication without affecting the viability of the host cell. Further detailed analyses indicate that 1-O-methyl-NBD-ceramide-C16 acts outside the inclusion. Thereby, 1-O-methyl-NBD-ceramide-C16 represents a lead compound for the development of novel anti-Chlamydia drugs and furthermore constitutes an agent to illuminate sphingolipid trafficking pathways in Chlamydia infections.

Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

The obligate intracellular pathogen Chlamydia trachomatis replicates within a membrane-bound inclusion that acquires host sphingomyelin (SM), a process that is essential for replication as well as inclusion biogenesis. Previous studies demonstrate that SM is acquired by a Brefeldin A (BFA)-sensitive vesicular trafficking pathway, although paradoxically, this pathway is dispensable for bacterial replication. This finding suggests that other lipid transport mechanisms are involved in the acquisition of host SM. In this work, we interrogated the role of specific components of BFA-sensitive and BFA-insensitive lipid trafficking pathways to define their contribution in SM acquisition during infection. We found that C. trachomatis hijacks components of both vesicular and non-vesicular lipid trafficking pathways for SM acquisition but that the SM obtained from these separate pathways is being utilized by the pathogen in different ways. We show that C. trachomatis selectively co-opts only one of the three known BFA targets, GBF1, a regulator of Arf1-dependent vesicular trafficking within the early secretory pathway for vesicle-mediated SM acquisition. The Arf1/GBF1-dependent pathway of SM acquisition is essential for inclusion membrane growth and stability but is not required for bacterial replication. In contrast, we show that C. trachomatis co-opts CERT, a lipid transfer protein that is a key component in non-vesicular ER to trans-Golgi trafficking of ceramide (the precursor for SM), for C. trachomatis replication. We demonstrate that C. trachomatis recruits CERT, its ER binding partner, VAP-A, and SM synthases, SMS1 and SMS2, to the inclusion and propose that these proteins establish an on-site SM biosynthetic factory at or near the inclusion. We hypothesize that SM acquired by CERT-dependent transport of ceramide and subsequent conversion to SM is necessary for C. trachomatis replication whereas SM acquired by the GBF1-dependent pathway is essential for inclusion growth and stability. Our results reveal a novel mechanism by which an intracellular pathogen redirects SM biosynthesis to its replicative niche.

HIV-1 does not significantly influence Chlamydia trachomatis serovar L2 replication in vitro

Individuals with lymphogranuloma venereum (LGV), caused by Chlamydia trachomatis serovar L2, are commonly co-infected with human immunodeficiency virus type 1 (HIV-1), for reasons that remain unknown. One hypothesis is that a biological synergy exists between the two pathogens. We tested this by characterising for the first time in vitro C. trachomatis L2 replication in the presence of HIV-1. The human epithelial cell-line, MAGI P4R5 was infected with C. trachomatis L2 and HIV-1 (MN strain). Co-infected cultures contained fewer and larger chlamydial inclusions, but the inclusions did not contain morphologically aberrant organisms. C. trachomatis remained infectious in the presence of HIV-1 and showed neither an alteration in genome accumulation, nor in the acumulation of ompA, euo or unprocessed 16S rRNA transcripts. However, omcB was slightly elevated. Taken together, these data indicate that HIV-1 co-infection did not significantly alter C. trachomatis replication and the association between HIV-1 and LGV is likely due to other factors that require further investigation. The fewer, larger inclusions observed in co-infected cultures probably result from the fusion of multiple inclusions in HIV-1 induced syncytia and indicate that C. trachomatis-host-cell interactions continue to function, despite considerable host-cell re-modelling.

CPLA2 Regulates the Expression of Type I Interferons and Intracellular Immunity to Chlamydia trachomatis

Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase RAS and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA(2) are activated and are important for the onset of inflammatory responses. In this study we tested if activation of ERK and cPLA(2) occurred as a result of RAS signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. We provide genetic and pharmacological evidence that during infection RAS, ERK, and, to a lesser extent, cPLA(2) activation are uncoupled, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA(2) signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells, inhibition of ERK and cPLA(2) played a significant protective role against C. trachomatis. We determined that cPLA(2)-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of beta interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Furthermore, the MAPK p38 was primarily responsible for cPLA(2) activation in Chlamydia-infected cells and IRG expression. Overall, these findings define a previously unrecognized role for cPLA(2) in the induction of cell autonomous cellular immunity to Chlamydia and highlight the many non-canonical signaling pathways engaged during infection.

The host adherens junction molecule nectin-1 is downregulated in Chlamydia trachomatis-infected genital epithelial cells

Nectin-1, a member of the immunoglobulin superfamily, is a Ca(2+)-independent cell adhesion protein implicated in the organization of E-cadherin-based adherens junctions (AJs) and claudin-based tight junctions (TJs) in epithelial cells. Nectin-1 also regulates cell-cell adhesion and cell polarization in a Cdc42- and Rac-dependent manner. Western blot analyses demonstrated that accumulation of host nectin-1 is decreased by 85 % at 48 hours post-infection (h.p.i.) in Chlamydia trachomatis serovar E-infected HeLa cells. Time-course experiments demonstrated that this decrease was sustained to 60 h.p.i. Nectin-1 downregulation in C. trachomatis-infected cells was prevented by both chloramphenicol exposure and prior inactivation of the chlamydiae with UV light, demonstrating that active C. trachomatis replication was required. Penicillin G-exposure studies demonstrated that nectin-1 accumulation was also altered during persistent infection. Finally, RT-PCR analyses indicated that chlamydial infection did not alter accumulation of any nectin-1 transcripts, demonstrating that nectin-1 accumulation is reduced at a post-transcriptional level. Intesrestingly, N-cadherin-dependent cell-cell junctions can be disrupted by C. trachomatis infection, as reported by Prozialeck et al. (2002). Because interaction of nectin molecules on adjacent cells is essential for AJ formation, these data suggest that C. trachomatis may disrupt AJs, at least in part, by diminishing nectin-1 accumulation. Notably, release of chlamydiae-infected epithelial cells has been observed both in vitro from polarized monolayers and in vivo from tissues, suggesting that chlamydia-modulated downregulation of adhesion molecules and the subsequent disruption of host cell adherence may be involved in chlamydial dissemination or pathogenesis.

Chlamydia muridarum Evades Growth Restriction by the IFN-γ-Inducible Host Resistance Factor Irgb10

Chlamydiae are obligate intracellular bacterial pathogens that exhibit a broad range of host tropism. Differences in host tropism between Chlamydia species have been linked to host variations in IFN-gamma-mediated immune responses. In mouse cells, IFN-gamma can effectively restrict growth of the human pathogen Chlamydia trachomatis but fails to control growth of the closely related mouse pathogen Chlamydia muridarum. The ability of mouse cells to resist C. trachomatis replication is largely dependent on the induction of a family of IFN-gamma-inducible GTPases called immunity-related GTPases or IRGs. In this study we demonstrate that C. muridarum can specifically evade IRG-mediated host resistance. It has previously been suggested that C. muridarum inactivates the IRG protein Irga6 (Iigp1) to dampen the murine immune response. However, we show that Irga6 is dispensable for the control of C. trachomatis replication. Instead, an effective IFN-gamma response to C. trachomatis requires the IRG proteins Irgm1 (Lrg47), Irgm3 (Igtp), and Irgb10. Ectopic expression of Irgb10 in the absence of IFN-gamma is sufficient to reduce intracellular growth of C. trachomatis but fails to restrict growth of C. muridarum, indicating that C. muridarum can specifically evade Irgb10-driven host responses. Importantly, we find that Irgb10 protein intimately associates with inclusions harboring C. trachomatis but is absent from inclusions formed by C. muridarum. These data suggest that C. muridarum has evolved a mechanism to escape the murine IFN-gamma response by restricting access of Irgb10 and possibly other IRG proteins to the inclusion.

Herpes simplex virus type 2 modulates the susceptibility of human bladder cells to uropathogenic bacteria.

The present study analyses the susceptibility of human bladder-derived cells (HT-1376) to the infection by herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis, as well as to the adhesiveness of uropathogenic bacteria. HT-1376 cells were efficiently infected by HSV-2 strain 333, as demonstrated by immunofluorescence staining of viral antigens, titration of cytopathic effect, and visualisation by transmission electron microscopy. This cell model was also prone to C. trachomatis (serovar E, Bour strain) replication and to the adherence of clinical uropathogenic isolates of Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Enterococcus faecalis. The pre-infection of HT-1376 cells with HSV-2 caused a tenfold increased adherence of an E. coli strain (U1), isolated from a patient affected by severe haemorrhagic cystitis, whereas in HSV-2 pre-infected cells the number of C. trachomatis inclusion bodies was significantly reduced. Our findings indicate that these cells are a suitable in vitro model for studying infection and super-infection of the lower urinary tract by viruses and bacteria.

Phospholipid composition of purified Chlamydia trachomatis mimics that of the eucaryotic host cell.

Chlamydia trachomatis is an obligate intracellular eubacterial parasite capable of infecting a wide range of eucaryotic host cells. Purified chlamydiae contain several lipids typically found in eucaryotes, and it has been established that eucaryotic lipids are transported from the host cell to the parasite. In this report, we examine the phospholipid composition of C. trachomatis purified from host cells grown under a variety of conditions in which the cellular phospholipid composition was altered. A mutant CHO cell line, with a thermolabile CDP-choline synthetase, was used to show that decreased host cell phosphatidylcholine levels had no significant effect on C. trachomatis growth. However, less phosphatidylcholine was transported to the parasite and purified elementary bodies contained decreased levels of phosphatidylcholine. Brefeldin A, fumonisin B1, and exogenous sphingomyelinase were used to alter levels of host cell sphingomyelin. None of the agents had a significant effect on C. trachomatis replication. Treatment with fumonisin B1 and exogenous sphingomyelinase resulted in decreased levels of host cell sphingomyelin. This had no effect on glycerophospholipid trafficking to chlamydiae; however, sphingomyelin trafficking was reduced and elementary bodies purified from treated cells had reduced sphingomyelin content. Exposure to brefeldin A, which had no adverse effect on chlamydia growth, resulted in an increase in cellular levels of sphingomyelin and a concomitant increase in the amount of sphingomyelin in purified chlamydiae. Under the experimental conditions used, brefeldin A treatment had only a small effect on sphingomyelin trafficking to the host cell surface or to C. trachomatis. Thus, the final phospholipid composition of purified C. trachomatis mimics that of the host cell in which it is grown.

Nitric oxide production: a mechanism of Chlamydia trachomatis inhibition in interferon-γ-treated RAW264.7 cells

IFN-γ and/or LPS induced nitrite production and inhibition of Chlamydia trachomatis (CT) replication in the murine macrophage cell line, RAW264.7. Linear regression analysis demonstrated a strong correlation between nitrite production and inhibition of CT replication (correlation coefficients: −0.93, P < 0.001). l-NMMA specifically inhibited nitrite production and restored CT replication (55–71%). Inducible nitric oxide synthase (iNOS) mRNA was analyzed by Northern and dot blot hybridization with an iNOS cDNA probe. A strong correlation between iNOS mRNA expression and inhibition of CT replication also was observed (correlation coefficient: −0.97, P < 0.05). Furthermore, anti-TNF-α antibody, which completely neutralized biological activity of the secreted TNF-α, neither inhibited nitrite production nor restored CT replication in the LPS- and/or IFN-γ-treated RAW264.7 cells. In mouse peritoneal macrophages treated with IFN-γ, both l-NMMA and anti-TNF-α antibody inhibited nitrite production and restored CT replication. However, l-NMMA and the antibody had no effect upon nitrite production and CT inhibition in LPS-treated peritoneal macrophages. These data indicate that NO production is one mechanism for inhibition of CT replication in IFN-γ-activated murine macrophages.

Nitric oxide production: a mechanism of Chlamydia trachomatis inhibition in interferon-gamma-treated RAW264.7 cells.

IFN-gamma and/or LPS induced nitrite production and inhibition of Chlamydia trachomatis (CT) replication in the murine macrophage cell line, RAW264.7. Linear regression analysis demonstrated a strong correlation between nitrite production and inhibition of CT replication (correlation coefficients: -0.93, P < 0.001). L-NMMA specifically inhibited nitrite production and restored CT replication (55-71%). Inducible nitric oxide synthase (iNOS) mRNA was analyzed by Northern and dot blot hybridization with an iNOS cDNA probe. A strong correlation between iNOS mRNA expression and inhibition of CT replication also was observed (correlation coefficient: -0.97, P < 0.05). Furthermore, anti-TNF-alpha antibody, which completely neutralized biological activity of the secreted TNF-alpha, neither inhibited nitrite production nor restored CT replication in the LPS- and/or IFN-gamma-treated RAW264.7 cells. In mouse peritoneal macrophages treated with IFN-gamma, both L-NMMA and anti-TNF-alpha antibody inhibited nitrite production and restored CT replication. However, L-NMMA and the antibody had no effect upon nitrite production and CT inhibition in LPS-treated peritoneal macrophages. These data indicate that NO production is one mechanism for inhibition of CT replication in IFN-gamma-activated murine macrophages.

In vitro synergy of clindamycin and aminoglycosides against Chlamydia trachomatis

The importance of Chlamydia trachomatis as an etiologic agent in the development of pelvic inflammatory disease (PID) is well documented. Although there are numerous antimicrobial agents that are effective against C. trachomatis, one of the most frequent combinations that is used to treat PID is clindamycin and gentamicin. The efficacy of clindamycin as the sole treatment for chlamydial infections has been questioned. In fact, the Centers for Disease Control (Atlanta, Ga.) has recommended the use of doxycycline following clindamycin and gentamicin treatment of PID confirmed or suspected to be caused by C. trachomatis. This study was designed to determine whether there is any synergistic in vitro activity between clindamycin and gentamicin or tobramycin on inhibition of C. trachomatis replication. In this experiment, the MIC of clindamycin decreased two- to threefold when an aminoglycoside was added. This occurred even though aminoglycosides by themselves had essentially no effect against C. trachomatis. The mechanism of this interaction is uncertain.

Inhibition of Chlamydia trachomatis replication in HEp-2 cells by human monocyte-derived macrophages.

Monocytes (M) and macrophages are important components of the immune response to foreign agents. Using an in vitro system, we studied the influence of human M and M-derived macrophages (MdM) on the replication of Chlamydia trachomatis (L2/434) in HEp-2 cells. M or MdM were added to infected cells at a ratio of 4:1, and the resultant chlamydial yield was evaluated in one-step growth experiments. Chlamydial DNA production was evaluated by dot hybridization. Both M and MdM reduced chlamydial yield and DNA production, but the reductions caused by MdM were more pronounced. Electron microscopy showed that while control HEp-2 cells at 48 h postinfection contained large inclusions in which most particles were elementary bodies, the infected HEp-2 cells exposed to MdM contained small vacuoles with abnormal reticulate bodies and very few typical elementary bodies. Separation of the MdM from the HEp-2 cells by a membrane reduced the inhibitory effect of the MdM relative to that of MdM in direct contact with the infected cells. Addition of tumor necrosis factor antibodies to C. trachomatis-infected HEp-2 cells exposed to MdM (either in direct contact or separated by a membrane from the infected cells) reduced the inhibition of chlamydial DNA production. These data suggest the possibility that MdM may modulate C. trachomatis replication in vivo.