NF-kappa B activation is not required for Chlamydia trachomatis inhibition of host epithelial cell apoptosis

Chlamydia trachomatis, an obligate intracellular bacterial species, is known to inhibit host cell apoptosis. However, the chlamydial antiapoptotic mechanism is still not clear. Because NF-kappaB activation is antiapoptotic, we tested the potential role of NF-kappaB activation in chlamydial antiapoptotic activity in the current study. First, no obvious NF-kappaB activation was detected in the chlamydia-infected cells when these cells were resistant to apoptosis induced via either the intrinsic or extrinsic apoptosis pathways. Second, inhibition of NF-kappaB activation with pharmacologic reagents failed to block the chlamydial antiapoptotic activity. Finally, NF-kappaB p65 gene deletion did not prevent chlamydia from inhibiting host cell apoptosis. These observations together have demonstrated that NF-kappaB activation is not required for the chlamydial antiapoptotic activity.     

Centrosome abnormalities during a Chlamydia trachomatis infection are caused by dysregulation of the normal duplication pathway

The presence of supernumerary centrosomes in cells infected with Chlamydia trachomatis may provide a mechanism to explain the association of C. trachomatis genital infection with cervical cancer. We show that the amplified centrosomal foci induced during a chlamydial infection contain both centriolar and pericentriolar matrix markers, demonstrating that they are bona fide centrosomes. As there were multiple immature centrioles but approximately one mature centriole per cell, aborted cytokinesis alone cannot account for centrosome amplification during a chlamydial infection. Production of supernumerary centrosomes required the kinase activities of Cdk2 and Plk4, which are known regulators of centrosome duplication, and progression through S-phase, which is the stage in the cell cycle when duplication of the centrosome occurs. These requirements indicate that centrosome amplification during a chlamydial infection depends on the host centrosome duplication pathway, which normally produces a single procentriole from each template centriole. However, C. trachomatis induces a loss of numerical control so that multiple procentrioles are formed per template.     

The role of intracellular glutathione in the progression of Chlamydia trachomatis infection

The productive internalization in the host cell of Chlamydia trachomatis elementary bodies and their infectivity depends on the degree of reduction of disulfide bonds in the outer envelope of the elementary body. We have hypothesized that the reducing agent may be intracellular glutathione (GSH). Three approaches were used to modulate the intracellular GSH concentration: (1) treatment of cells with buthionine sulfoximine, which causes irreversible inhibition of GSH biosynthesis; (2) hydrogen peroxide-induced oxidation of GSH by intracellular glutathione peroxidases; and (3) treatment of cells with N-acetyl-l-cysteine (NAC), a precursor of glutathione. In the first two cases, we observed a four- to sixfold inhibition of C. trachomatis infection, whereas in NAC-treated cells we detected an increase in the size of chlamydial inclusions. Using a proteomics approach, we showed that the inhibition of chlamydial infection does not combine with alterations in protein expression patterns after cell treatment. These results suggest that GSH plays a key role in the reduction of disulfide bonds in the C. trachomatis outer envelope at an initial stage of the infection.     

Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia -containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule-associated Chlamydia vesicles also collocated with tyrosine-phosphorylated McCoy cell proteins. After migration, the Chlamydia -containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule-dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule-dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR-1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR-1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis .     

Type I IFNs enhance susceptibility to Chlamydia muridarum lung infection by enhancing apoptosis of local macrophages

Type I IFNs (IFNIs) have pleiotropic functions in regulating host innate and adaptive immune responses to pathogens. To elucidate the role of IFNIs in host resistance to chlamydial infection in vivo, we compared IFN-alpha/beta receptor knockout (IFNAR(-/-)) and wild-type control mice in susceptibility to Chlamydia trachomatis mouse pneumonitis (Chlamydia muridarum) lung infection. We found that the IFNAR(-/-) mice were significantly more resistant to C. muridarum infection showing less bacterial burden and bodyweight loss, and milder pathological changes. However, IFN-gamma response, which is believed to be critical in host defense against chlamydial infection, was similar between the wild-type and IFNAR(-/-) mice. More importantly, TUNEL analysis showed less macrophage apoptosis in IFNAR(-/-) mice, which was consistent with lower expressions of IFNI-induced apoptotic factors, TRAIL, Daxx, and PKR. Furthermore, depletion of lung macrophages with dichloromethylene diphosphonate-liposome significantly increased the susceptibility of the IFNAR(-/-) mice to C. muridarum, confirming the importance of macrophages. Overall, the data indicate that IFNIs play a promoting role in C. muridarum lung infection, largely through increase of local macrophage apoptosis.     

Interaction of Chlamydia trachomatis with human genital epithelium in culture

Primary cultures of human endometrial and ectocervical epithelial cells were examined as a new model system to study genital infection by Chlamydia trachomatis. Initial studies demonstrated that these cells were indeed susceptible to chlamydial infection. Inocula, adjusted to produce inclusions in 50 to 80% of equivalent numbers of standard McCoy cells, resulted in infection rates of approximately 15 to 30% for the columnar cells of the endometrium and 5 to 10% for the squamous cells of the ectocervix. Exposure of cultures to DEAE-dextran and centrifugation-assisted inoculation, manipulations reported to enhance infection of HeLa and McCoy cells, did not alter the number of inclusion-positive genital cells. Addition of cycloheximide to the post-inoculation culture medium slightly increased numbers of inclusion-bearing cells while growth of genital cells in hormone-supplemented medium resulted in a variable effect on inclusion development and a significant reduction in the association of radiolabelled organisms with these cells. The basis for the different levels of infection in McCoy versus genital cell cultures was revealed by immunofluorescence analysis of chlamydial association with host cells immediately after inoculation. Chlamydiae failed to adhere to many cells in the genital cell cultures while adherence to McCoy cells was uniform. In addition, the association of radiolabelled C. trachomatis was significantly lower with genital cells than with McCoy cells. Finally, culture conditions were defined which markedly inhibited inclusion development without an immediate loss of chlamydial growth potential. This investigation indicates that primary genital cell cultures are susceptible to chlamydial infection and will be valuable for studies on the nature of C. trachomatis interactions with natural human target cells.     

Effect of 6-thioguanine on Chlamydia trachomatis growth in wild-type and hypoxanthine-guanine phosphoribosyltransferase-deficient cells.

Chlamydiae have evolved a biphasic life cycle to facilitate their survival in two discontinuous habitats. The unique growth cycle is represented by two alternating forms of the organism, the elementary body and the reticulate body. Chlamydiae have an absolute nutritional dependency on the host cell to provide ribonucleoside triphosphates and other essential intermediates of metabolism. This report describes the pleiotropic effects of the purine antimetabolite 6-thioguanine on chlamydial replication. In order to display cytotoxicity, 6-thioguanine must first be converted to the nucleotide level by the host cell enzyme hypoxanthine-guanine phosphoribosyltransferase. Our results show that 6-thioguanine is an effective inhibitor of chlamydial growth with either wild-type or hypoxanthine-guanine phosphoribosyltransferase-deficient cell lines as the host. Interestingly, the mechanism of 6-thioguanine-induced inhibition of chlamydial growth is different depending on which cell line is used. With wild-type cells as the host, the cytotoxic effects of 6-thioguanine on chlamydial growth are relatively fast and irreversible. Under these circumstances, cytotoxicity likely results from the combined effect of starving chlamydiae for purine ribonucleotides and incorporation of host-derived 6-thioguanine-containing nucleotides into chlamydial nucleic acids. With hypoxanthine-guanine phosphoribosyltransferase-deficient cells as the host, 6-thioguanine must be present at the start of the chlamydial infection cycle to be effective and the growth inhibition is reversible upon removal of the antimetabolite. These findings suggest that in hypoxanthine-guanine phosphoribosyltransferase-deficient cells, the free base 6-thioguanine may inhibit the differentiation of elementary bodies to reticulate bodies. With hypoxanthine-guanine phosphoribosyltransferase-deficient cells as the host, 6-thioguanine was used as a selective agent in culture to isolate a Chlamydia trachomatis isolate resistant to the effects of the drug. This drug resistant C. trachomatis isolate was completely resistant to 6-thioguanine in hypoxanthine-guanine phosphoribosyltransferase-deficient cells; however, it displayed wildtype sensitivity to 6-thioguanine when cultured in wild-type host cells.     

Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector

Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by complementation.     

Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle

The obligate intracellular bacterium Chlamydia trachomatis possesses a biphasic developmental cycle that is manifested by differentiation of infectious, metabolically inert elementary bodies (EBs) to larger, metabolically active reticulate bodies (RBs). The cycle is completed by asynchronous differentiation of dividing RBs back to a population of dormant EBs that can initiate further rounds of infection upon lysis of the host cell. Chlamydiae express a type III secretion system (T3SS) that is presumably employed to establish and maintain the permissive intracellular niche by secretion of anti-host proteins. We hypothesize that T3SS activity is essential for chlamydial development and pathogenesis. However, the lack of a genetic system has confounded efforts to establish any role of the T3SS. We therefore employed the small molecule Yersinia T3SS inhibitor N'-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide, designated compound 1 (C1), to examine the interdependence of the chlamydial T3SS and development. C1 treatment inhibited C. trachomatis but not T4SS-expressing Coxiella burnetii development in a dose-dependent manner. Although chlamydiae remained viable and metabolically active, they failed to divide significantly and RB to EB differentiation was inhibited. These effects occurred in the absence of host cell cytotoxicity and were reversible by washing out C1. We further demonstrate that secretion of T3S substrates is perturbed in C1-treated chlamydial cultures. We have therefore provided evidence that C1 can inhibit C. trachomatis development and T3SS activity and present a model in which progression of the C. trachomatis developmental cycle requires a fully functional T3SS.     

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

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

The multifaceted role of oestrogen in enhancing Chlamydia trachomatis infection in polarized human endometrial epithelial cells

The oestrogen receptor (ER) α-β+ HEC-1B and the ERα+β+ Ishikawa (IK) cell lines were investigated to dissect the effects of oestrogen exposure on several parameters of Chlamydia trachomatis infection. Antibody blockage of ERα or ERβ alone or simultaneously significantly decreased C. trachomatis infectivity (45-68%). Addition of the ERβ antagonist, tamoxifen, to IK or HEC-1B prior to or after chlamydial infection caused a 30-90% decrease in infectivity, the latter due to disrupted eukaryotic organelles. In vivo, endometrial glandular epithelial cells are stimulated by hormonally influenced stromal signals. Accordingly, chlamydial infectivity was significantly increased by 27% and 21% in IK and HEC-1B cells co-cultured with SHT-290 stromal cells exposed to oestrogen. Endometrial stromal cell/epithelial cell co-culture revealed indirect effects of oestrogen on phosphorylation of extracellular signal-regulated kinase and calcium-dependant phospholipase A2 and significantly increased production of interleukin (IL)-8 and IL-6 in both uninfected and chlamydiae-infected epithelial cells. These results indicate that oestrogen and its receptors play multiple roles in chlamydial infection: (i) membrane oestrogen receptors (mERs) aid in chlamydial entry into host cells, and (ii) mER signalling may contribute to inclusion development during infection. Additionally, enhancement of chlamydial infection is affected by hormonally influenced stromal signals in conjunction with direct oestrogen stimulation of the human epithelia.     

Chlamydia heat shock protein 60 induces trophoblast apoptosis through TLR4

Intrauterine infection affects placental development and function, and subsequently may lead to complications such as preterm delivery, intrauterine growth retardation, and preeclampsia; however, the molecular mechanisms are not clearly known. TLRs mediate innate immune responses in placenta, and recently, TLR2-induced trophoblast apoptosis has been suggested to play a role in infection-induced preterm delivery. Chlamydia trachomatis is the etiological agent of the most prevalent sexually transmitted bacterial infection in the United States. In this study, we show that in vitro chlamydial heat shock protein 60 induces apoptosis in primary human trophoblasts, placental fibroblasts, and the JEG3 trophoblast cell line, and that TLR4 mediates this event. We observed a host cell type-dependent apoptotic response. In primary placental fibroblasts, chlamydial heat shock protein 60-induced apoptosis was caspase dependent, whereas in JEG3 trophoblast cell lines it was caspase independent. These data suggest that TLR4 stimulation induces apoptosis in placenta, and this could provide a novel mechanism of pathogenesis for poor fertility and pregnancy outcome in women with persistent chlamydia infection.     

Chlamydia trachomatis infection of human fallopian tube organ cultures

The pathogenic events that precede Chlamydia trachomatis salpingitis in the human fallopian tube have not been fully described. We used a model of human fallopian tubes in organ culture (HFTOC) infected with strain E/UW-5/CX of C. trachomatis to study these events. The model supported sustained C. trachomatis infection as demonstrated by recovery of viable C. trachomatis from medium and tissue over 5-7 d. However, the level of infectivity was low. Maximal infection occurred at 72 h after initial inoculation. In contrast to gonococcal infection of the HFTOC, C. trachomatis did not damage overall ciliary function of HFTOC. However, a local direct cytotoxic effect characterized by loss of microvilli and disruption of cell junctions was noted when multiple chlamydial elementary bodies attached to mucosal cells. Beginning at 24 h, and continuing throughout the course of C. trachomatis infection of HFTOC, ruptured epithelial cells releasing elementary bodies were noted. Chlamydial inclusions were seen in the mucosa by 72 h in approximately 6% of both ciliated and nonciliated epithelial cells. Mucosal inclusions contained all forms of the C. trachomatis developmental cycle. These data suggest that factors present in the human fallopian tube may limit susceptibility to chlamydial infection but support the use of the HFTOC model in the study of the pathogenesis of C. trachomatis salpingitis.     

Lysosome repair enables host cell survival and bacterial persistence following Chlamydia trachomatis infection

Chlamydiae are obligate intracellular bacteria that replicate within the confines of a membrane-bound vacuole termed the inclusion. The final event in the infectious process is the disruption of the inclusion membrane and release of a multitude of infectious elementary bodies, each capable of eliciting a new infection. Strains of the trachoma biovar of Chlamydia trachomatis are released from the host cell without concomitant host cell death. In this study, analysis of events associated with chlamydial egress revealed that the integrity of the host cell plasma membrane was compromised prior to the inclusion membrane. This disruption was accompanied by the appearance of LAMP-1 at the infected cell surface, implicating lysosome repair of plasma membrane lesions in response to infection. Analysis of the effects of calcium chelators and actin stabilizing agents, indicated calcium-induced actin depolymerization as a requisite to lysosome-plasma membrane fusion and host cell survival. A consequence of this lysosome-mediated repair process, was the retention of residual bacteria within the surviving host cell, providing a unique mechanism for intracellular persistence of C. trachomatis.     

Reduced display of tumor necrosis factor receptor I at the host cell surface supports infection with Chlamydia trachomatis

The obligate intracellular human pathogenic bacterium Chlamydia trachomatis has evolved multiple mechanisms to circumvent the host immune system. Infected cells exhibit a profound resistance to the induction of apoptosis and down-regulate the expression of major histocompatibility complex class I and class II molecules to evade the cytotoxic effect of effector immune cells. Here we demonstrate the down-regulation of tumor necrosis factor receptor 1 (TNFR1) on the surface of infected cells. Interestingly, other members of the TNFR family such as TNFR2 and CD95 (Fas/Apo-1) were not modulated during infection, suggesting a selective mechanism underlying surface reduction of TNFR1. The observed effect was not due to reduced expression since the overall amount of TNFR1 protein was increased in infected cells. TNFR1 accumulated at the chlamydial inclusion and was shed by the infected cell into the culture supernatant. Receptor shedding depended on the infection-induced activation of the MEK-ERK pathway and the metalloproteinase TACE (TNFalpha converting enzyme). Our results point to a new function of TNFR1 modulation by C. trachomatis in controlling inflammatory signals during infection.     

Host cell phospholipids are trafficked to and then modified by Chlamydia trachomatis.

There is little information on the trafficking of eukaryotic lipids from a host cell to either the cytoplasmic membrane of or the vacuolar membrane surrounding intracellular pathogens. Purified Chlamydia trachomatis, an obligate intracellular bacterial parasite, contains several eukaryotic glycerophospholipids, yet attempts to demonstrate transfer of these lipids to the chlamydial cell membrane have not been successful. In this report, we demonstrate that eukaryotic glycerophospholipids are trafficked from the host cell to C. trachomatis. Phospholipid trafficking was assessed by monitoring the incorporation of radiolabelled isoleucine, a precursor of C. trachomatis specific branched-chain fatty acids, into host-derived glycerophospholipids and by monitoring the transfer of host phosphatidylserine to chlamydiae and its subsequent decarboxylation to form phosphatidylethanolamine. Phospholipid trafficking to chlamydiae was unaffected by brefeldin A, an inhibitor of Golgi function. Furthermore, no changes in trafficking were observed when C. trachomatis was grown in a mutant cell line with a nonfunctional, nonspecific phospholipid transfer protein. Host glycerophospholipids are modified by C. trachomatis, such that a host-synthesized straight-chain fatty acid is replaced with a chlamydia-synthesized branched-chain fatty acid. We also demonstrate that despite the acquisition of host-derived phospholipids, C. trachomatis is capable of de novo synthesis of phospholipids typically synthesized by prokaryotic cells. Our results provide novel information on chlamydial phospholipid metabolism and eukaryotic cell lipid trafficking, and they increase our understanding of the evolutionary steps leading to the establishment of an intimate metabolic association between an obligate intracellular bacterial parasite and a eukaryotic host cell.