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Chlamydia muridarum and Chlamydia caviae have equivalent growth rates in mouse epithelial cells but only C. muridarum replicates inside mouse macrophages, while C. caviae does not. Macrophages infected with C. muridarum or C. caviae were used to address the hypothesis that the early signaling pathways initiated during infection depend on the fate of chlamydiae in the host cell. Transmission electron microscopy of C. muridarum-infected macrophages showed intact chlamydial elementary bodies and reticulate bodies 2 h postinfection in compact vacuoles. Conversely, in macrophages infected with C. caviae, chlamydiae were observed in large phagocytic vacuoles. Furthermore, C. caviae infections failed to develop into inclusions or produce viable bacteria. Expression of proinflammatory cytokines TNFα, IL-1β and MMP13 was similar in C. caviae- or C. muridarum-infected macrophages at 3 h postinfection, indicating that chlamydial survival is not required for initiation of these responses. IL-1β secretion, dependent on inflammasome activation, occurred in C. caviae-infected macrophages despite no chlamydial growth. Conversely, IFNβ mRNA was observed only in C. muridarum- but not in C. caviae-infected macrophages. These data demonstrate that differential signaling events are initiated during a productive versus nonproductive chlamydial infection in a macrophage., (© 2017 Australasian Society for Immunology Inc.)

Studies indicate that estrogen enhances Chlamydia trachomatis serovar E infection in genital epithelial cells. Hormones have direct and indirect effects on endometrial epithelial cells. Estrogen and progesterone exposure induces endometrial stromal cells to release effectors that subsequently regulate growth and maturation of uterine epithelial cells. Estrogen enhances C. trachomatis infection by aiding entry and intracellular development in endometrial epithelial cell (Ishikawa, IK)/SHT-290 stromal cell co-culture. Enhanced chlamydial infection was mediated by direct estrogen-stimulated signaling events in epithelial cells and indirectly via estrogen-induced stromal cell effectors. The current study investigates the effects of hormones on chlamydial development using culture conditions representative of the menstrual cycle. Chlamydia trachomatis-infected IK or IK/SHT-290 cultures were exposed to 10(-8) M estrogen (E2), 10(-7) M progesterone (P4) or a combination of both hormones (10(-8) M E2 followed by 10(-9) M E2/10(-7) M P4). Chlamydial infectivity and progeny production were significantly decreased (30-66%) in cultures exposed to progesterone or estrogen/progesterone combination compared to estrogen alone. Thus, progesterone antagonized the positive effects of estrogen on chlamydial infection. These data indicate the susceptibility of endometrial epithelial cells to C. trachomatis infection during the menstrual cycle is altered by phase specific actions of sex hormones in the genital tract., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

IFN-β has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFN-β expression during chlamydial infection. We determined that three-prime repair exonuclease-1, a host 3' to 5' exonuclease, reduced IFN-β expression significantly during chlamydial infection using small interfering RNA and gene knockout fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cyclic GMP-AMP synthase (cGAS) has been shown to bind cytosolic DNA to generate cyclic GMP-AMP, which binds to the signaling adaptor stimulator of IFN genes (STING) to induce IFN-β expression. We determined that cGAS is required for IFN-β expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFN-β, coculture of cells depleted for either STING or cGAS rescued IFN-β expression. These data demonstrate that cyclic GMP-AMP produced in infected cGAS(+)STING(-) cells can migrate into adjacent cells via gap junctions to function in trans in cGAS(-)STING(+) cells. Furthermore, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is most likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated DNA sensing directs IFN-β expression during Chlamydia trachomatis infection and suggest that effectors from infected cells can directly upregulate IFN-β expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis., (Copyright © 2014 by The American Association of Immunologists, Inc.)

We utilized a recently developed model of intracervical infection with Chlamydia muridarum in the mouse to elicit a relatively synchronous infection during the initial developmental cycle in order to examine at the ultrastructural level the development of both the chlamydial inclusion and the onset of the inflammatory response. At 18 h after infection, only a few elementary bodies attached to cells were visible, as were an occasional intracellular intermediate body and reticulate body. By 24 h, inclusions had 2 to 5 reticulate bodies and were beginning to fuse. A few polymorphonuclear leukocytes (PMNs) were already present in the epithelium in the vicinity of and directly adjacent to infected cells. By 30 h, the inclusions were larger and consisted solely of reticulate bodies, but by 36 to 42 h, they contained intermediate bodies and elementary bodies as well. Many PMNs were adjacent to or actually inside infected cells. Chlamydiae appeared to exit the cell either (i) through disintegration of the inclusion membrane and rupture of the cell, (ii) by dislodgement of the cell from the epithelium by PMNs, or (iii) by direct invasion of the infected cell by the PMNs. When PMNs were depleted, the number of released elementary bodies was significantly greater as determined both visually and by culture. Interestingly, depletion of PMNs revealed the presence of inclusions containing aberrant reticulate bodies, reminiscent of effects seen in vitro when chlamydiae are incubated with gamma interferon. In vivo evidence for the contact-dependent development hypothesis, a potential mechanism for triggering the conversion of reticulate bodies to elementary bodies, and for translocation of lipid droplets into the inclusion is also presented.

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., (© 2011 Blackwell Publishing Ltd.)

Chlamydiae growing in target mucosal human epithelial cells in vitro can transition from their normal developmental cycle progression, alternating between infectious but metabolically inactive elementary bodies to metabolically active but noninfectious reticulate bodies (RBs) and back to elementary bodies, into a state of persistence. Persistence in vitro is defined as viable but noncultivable chlamydiae involving morphologically enlarged, aberrant, and nondividing RBs. The condition is reversible, yielding infectious elementary bodies after removal of the inducers, including penicillin, interferon-gamma, iron or nutrient starvation, concomitant herpes infection, or maturation of the host cell into its physiologically differentiated state. All aberrant RB phenotypes are not the same, owing to differing up- or down-regulated chlamydial gene sets and subsequent host responses. Although all persistence-inducing conditions exist in vivo, key questions include (1) whether or not aberrant chlamydial RBs occur in vivo during the alternating acute-silent chronic-acute chlamydial infection scenario that exists in infected patients and animals and (2) whether such aberrant RBs can contribute to prolonged, chronic inflammation, fibrosis, and scarring.

The initial host response in a primary chlamydial infection is the onset of acute inflammation. However, we still know very little about the early temporal events in the induction of the acute inflammatory response and how these events relate to the initial chlamydial developmental cycle in an actual genital infection. Because it was critical to initiate a synchronous infection in the endocervix in the first 24 h to evaluate the sequential expression of the host response, we developed the surgical methodology of depositing Chlamydia muridarum directly on the endocervix. Cervical tissue was collected at 3, 12, and 24 h after inoculation and the expression array of chemokines, cytokines, and receptors was assessed to characterize the response during the initial developmental cycle. Polymorphonuclear leukocyte (PMN) infiltration was first observed at 12 h after inoculation, and a few PMNs could be seen in the epithelium at 24 h. Electron microscopic analysis at 24 h showed that virtually all inclusions were at the same stage of development, indicating a synchronous infection. Several chemokine and cytokine genes were expressed as early as 3 h after infection, but by 12 h, 41 genes were expressed. Thus, activation of the host response occurs both with the introduction of elementary bodies into the host and early replication of reticulate bodies. No significant response was observed when UV-inactivated organisms were inoculated into the cervix at any time interval. This model provides an ideal opportunity to investigate the mechanisms by which the early inflammatory response is induced in vivo.

Confinement of the obligate intracellular bacterium Chlamydia trachomatis to a membrane-bound vacuole, termed an inclusion, within infected epithelial cells neither prevents secretion of chlamydial antigens into the host cytosol nor protects chlamydiae from innate immune detection. However, the details leading to chlamydial antigen presentation are not clear. By immunoelectron microscopy of infected endometrial epithelial cells and in isolated cell secretory compartments, chlamydial major outer membrane protein (MOMP), lipopolysaccharide (LPS) and the inclusion membrane protein A (IncA) were localized to the endoplasmic reticulum (ER) and co-localized with multiple ER markers, but not with markers of the endosomes, lysosomes, Golgi nor mitochondria. Chlamydial LPS was also co-localized with CD1d in the ER. Since the chlamydial antigens, contained in everted inclusion membrane vesicles, were found within the host cell ER, these data raise additional implications for antigen processing by infected uterine epithelial cells for classical and non-classical T cell antigen presentation.

While much is known about the attachment of the chlamydiae to the host cell and intracellular events during the developmental cycle, little is known about the mechanism(s) by which elementary bodies exit the cell. In this report, we use the guinea-pig conjunctival model of Chlamydia caviae infection to present in vivo ultrastructural evidence supporting two mechanisms for release of chlamydiae from the mucosal epithelia. Four days after infection, histopathologic observation shows an intense infiltration of polymorphonuclear leukocytes (PMN) in the conjunctival epithelium. Using transmission electron microscopy, a gradient-directed PMN response to chlamydiae-infected epithelial cells was observed. As PMN infiltration intensifies, epithelial hemidesmosome/integrin/focal adhesion adherence with the basal lamina is disconnected and PMNs literally lift off and release infected superficial epithelia from the mucosa. Many of these infected cells appear to be healthy with intact microvilli, nuclei, and mitochondria. While lysis of some infected cells occurs with release of chlamydiae into the extracellular surface milieu, the majority of infected cells are pushed off the epithelium. We propose that PMNs play an active role in detaching infected cells from the epithelium and that these infected cells eventually die releasing organisms but, in the process, move to new tissue sites via fluid dynamics.

A common model for studying Chlamydia trachomatis and growing chlamydial stocks uses Lymphogranuloma venereum serovar L2 and non-polarized HeLa cells. However, recent publications indicate that the growth rate and progeny yields can vary considerably for a particular strain depending on the cell line/type used, and seem to be partially related to cell tropism. In the present study, the growth of invasive serovar L2 was compared in endometrial HEC-1B and endocervical HeLa cells polarized on collagen-coated microcarrier beads, as well as in HeLa cells grown in tissue culture flasks. Microscopy analysis revealed no difference in chlamydial attachment/entry patterns or in inclusion development throughout the developmental cycle between cell lines. Very comparable growth curves in both cell lines were also found using real-time PCR analysis, with increases in chlamydial DNA content of 400-500-fold between 2 and 36 h post-inoculation. Similar progeny yields with comparable infectivity were recovered from HEC-1B and HeLa cell bead cultures, and no difference in chlamydial growth was found in polarized vs. non-polarized HeLa cells. In conclusion, unlike other C. trachomatis strains such as urogenital serovar E, invasive serovar L2 grows equally well in physiologically different endometrial and endocervical environments, regardless of the host cell polarization state.

Chlamydia spp. express a functional type III secretion system (T3SS) necessary for pathogenesis and intracellular growth. However, certain essential components of the secretion apparatus have diverged to such a degree as to preclude their identification by standard homology searches of primary protein sequences. One example is the needle subunit protein. Electron micrographs indicate that chlamydiae possess needle filaments, and yet database searches fail to identify a SctF homologue. We used a bioinformatics approach to identify a likely needle subunit protein for Chlamydia. Experimental evidence indicates that this protein, designated CdsF, has properties consistent with it being the major needle subunit protein. CdsF is concentrated in the outer membrane of elementary bodies and is surface exposed as a component of an extracellular needle-like projection. During infection CdsF is detectable by indirect immunofluorescence in the inclusion membrane with a punctuate distribution adjacent to membrane-associated reticulate bodies. Biochemical cross-linking studies revealed that, like other SctF proteins, CdsF is able to polymerize into multisubunit complexes. Furthermore, we identified two chaperones for CdsF, termed CdsE and CdsG, which have many characteristics of the Pseudomonas spp. needle chaperones PscE and PscG, respectively. In aggregate, our data are consistent with CdsF representing at least one component of the extended Chlamydia T3SS injectisome. The identification of this secretion system component is essential for studies involving ectopic reconstitution of the Chlamydia T3SS. Moreover, we anticipate that CdsF could serve as an efficacious target for anti-Chlamydia neutralizing antibodies.

In vitro studies of obligate intracellular chlamydia biology and pathogenesis are highly dependent on the use of experimental models and growth conditions that mimic the mucosal architecture and environment these pathogens encounter during natural infections. In this study, the growth of Chlamydia trachomatis genital serovar E was monitored in mouse fibroblast McCoy cells and compared to more relevant host human epithelial endometrium-derived HEC-1B and cervix-derived HeLa cells, seeded and polarized on collagen-coated microcarrier beads, using a three-dimensional culture system. Microscopy analysis of these cell lines prior to infection revealed morphological differences reminiscent of their in vivo architecture. Upon infection, early chlamydial inclusion distribution was uniform in McCoy cells but patchy in both epithelial cell lines. Although no difference in chlamydial attachment to or entry into the two genital epithelial cell lines was noted, active bacterial genome replication and transcription, as well as initial transformation of elementary bodies to reticulate bodies, were detected earlier in HEC-1B than in HeLa cells, suggesting a faster growth, which led to higher progeny counts and titers in HEC-1B cells upon completion of the developmental cycle. Chlamydial development in the less relevant McCoy cells was very similar to that in HeLa cells, although higher progeny counts were obtained. In conclusion, this three-dimensional bead culture system represents an improved model for harvesting large quantities of infectious chlamydia progeny from their more natural polarized epithelial host cells.

Modification of the phosphate groups of lipid A with amine-containing substituents, such as phosphoethanolamine, reduces the overall net negative charge of gram-negative bacterial lipopolysaccharide, thereby lowering its affinity to cationic antimicrobial peptides. Modification of the 1 position of Helicobacter pylori lipid A is a two-step process involving the removal of the 1-phosphate group by a lipid A phosphatase, LpxEHP (Hp0021), followed by the addition of a phosphoethanolamine residue catalyzed by EptAHP (Hp0022). To demonstrate the importance of modifying the 1 position of H. pylori lipid A, we generated LpxEHP-deficient mutants in various H. pylori strains by insertion of a chloramphenicol resistance cassette into lpxEHP and examined the significance of LpxE with respect to cationic antimicrobial peptide resistance. Using both mass spectrometry analysis and an in vitro assay system, we showed that the loss of LpxEHP activity in various H. pylori strains resulted in the loss of modification of the 1 position of H. pylori lipid A, thus confirming the function of LpxEHP. Due to its unique lipid A structure, H. pylori is highly resistant to the antimicrobial peptide polymyxin (MIC > 250 microg/ml). However, disruption of lpxEHP in H. pylori results in a dramatic decrease in polymyxin resistance (MIC, 10 microg/ml). In conclusion, we have characterized the first gram-negative LpxE-deficient mutant and have shown the importance of modifying the 1 position of H. pylori lipid A for resistance to polymyxin.

Several chlamydial antigens have been detected in the infected epithelial cell cytosol and on the host cell surface prior to their presumed natural release at the end of the 72-96 h developmental cycle. These extra-inclusion antigens are proposed to influence vital host cell functions, antigen trafficking and presentation and, ultimately, contribute to a prolonged inflammatory response. To begin to dissect the mechanisms for escape of these antigens from the chlamydial inclusion, which are enhanced on exposure to antibiotics, polarized endometrial epithelial cells (HEC-1B) were infected with Chlamydia trachomatis serovar E for 36 h or 48 h. Infected cells were then exposed to chemotactic human polymorphonuclear neutrophils not loaded or pre-loaded in vitro with the antibiotic azithromycin. Viewed by electron microscopy, the azithromycin-mediated killing of chlamydiae involved an increase in chlamydial outer membrane blebbing followed by the appearance of the blebs in larger vesicles (i) everting from but still associated with the inclusion as well as (ii) external to the inclusion. Evidence that the vesicles originated from the chlamydial inclusion membrane was shown by immuno-localization of inclusion membrane proteins A, F, and G on the vesicular membranes. Chlamydial heat shock protein 60 (chsp60) copies 2 and 3, but not copy 1, were released from RB and incorporated into the everted inclusion membrane vesicles and delivered to the infected cell surface. These data represent direct evidence for one mechanism of early antigen delivery, albeit membrane-bound, beyond the confines of the chlamydial inclusion.

Epidemiological and clinical studies have shown that double infection with herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occurs in vivo. We hypothesized that co-infection would alter replication of these agents. To test this hypothesis, HeLa cells were infected with C. trachomatis serovar E, followed 24 h later by HSV-2 strain 333. Transmission electron microscopic (TEM) analyses indicated that, by 10 h after HSV addition, reticulate bodies (RBs) in co-infected cells were swollen, aberrantly shaped and electron-lucent. In infectious titre assays, HSV-2 co-infection abrogated production of infectious chlamydial progeny. Western blot analyses indicated that accumulation of chlamydial major outer membrane protein (MOMP) was decreased by HSV co-infection while accumulation of chlamydial heat-shock protein 60-1 (HSP60-1) was increased. Polymerase chain reaction (PCR) experiments indicated that chlamydial genome copy number was unaltered by HSV-2 superinfection. Semi-quantitative, reverse transcription PCR (RT-PCR) experiments demonstrated that levels of chlamydial groEL, ftsK, ftsW, dnaA and unprocessed 16S rRNA transcripts were not changed by HSV-2 super-infection. These data indicate that HSV-2 superinfection drives chlamydia into a viable but non-cultivable state, which is the hallmark of persistence. Because chlamydial HSP60-1 has been associated with immunopathology in vivo, these results also suggest that disease severity might be increased in co-infected individuals.

Chlamydial attachment and infectivity in vitro and ascending disease and sequelae in vivo have been reported to be enhanced/modulated by estrogen. Endometrial carcinoma cell lines Ishikawa and HEC-1B and the breast cancer lines MCF-7 and HCC-1806 were examined for Chlamydia trachomatis E infectivity. Estrogen receptor (ER) presence was confirmed by Western blot and qRT-PCR analyses. FACS analysis was used to determine the percent of plasma membrane-localized ERs (mERs), and their activity was tested by estrogen binding and competitive estrogen antagonists assays. Chlamydiae grew in all cell lines with HEC (90%) >> MCF-7 (57%)>Ishikawa (51%) >> HCC-1806 (20%). The cell line ER isoform composition was re-defined as: ERalpha + ERbeta + for MCF-7, HCC-1806 and Ishikawa; and ERbeta only for HEC-1B. HeLa cells were also tested and found to express ERbeta, but not ERalpha. A small percentage of both ERs were surface-exposed and functionally active. The endometrium-predominant ERbeta isoform was found in all cell lines, including those most representative of the common sites of C. trachomatis infection. Thus, the role of chlamydial attachment/infectivity will now be analyzed in ERbeta+and-isogenic HEC-1B cells.

Objective: The clinical significance of the potential for persistent human chlamydial infections in vivo is being actively reassessed because of the increased frequency of recurrent infection with the same serovar despite compliance with an effective antibiotic regimen. The ability to extend the length of time of in vitro cultivation of polarized human endometrial epithelial cells (HEC-1B) provided the opportunity to establish a model system to determine if a persistent form of Chlamydia trachomatis had the same susceptibility as the actively growing form to a cidal concentration of azithromycin., Methods: Polarized HEC-1B cells cultivated on extracellular matrix were infected with C. trachomatis serovar E and exposed to penicillin at 24 h post-infection (hpi) to induce a persistent infection characterized by slowly metabolizing but non-dividing, ultrastructurally aberrant reticulate bodies within the chlamydial inclusion; at 48 hpi, infected cultures were exposed to a bactericidal concentration of azithromycin for 72 h., Results: Persistent chlamydiae were phenotypically resistant to azithromycin; the number of chlamydial inclusions on subpassage of progeny from persistent chlamydiae following removal of penicillin and recovery was essentially the same as from progeny from persistent chlamydiae following removal of penicillin and azithromycin and recovery. Neutrophils were attracted in vitro to persistently infected HEC-1B cells that had been exposed to penicillin and azithromycin., Conclusions: Thus, this study provides evidence at the cellular microbiology level in vitro for mechanisms that could exist in vivo to create sustained, but perhaps clinically inapparent inflammation, which might eventually lead to conditions such as silent pelvic inflammatory disease.

Causative molecular mechanisms accounting for the potential link between Chlamydia pneumoniae and atherosclerosis are unknown. Formalin and heat-inactivated C. pneumoniae activated the transcription factor nuclear factor (NF)-kappaB in cultured porcine endothelium and up-regulated the expression of E-selectin messenger RNA and protein. This up-regulation was abolished by an IkappaB super-repressor, an NF-kappaB-specific inhibitor. Live bacteria are not necessary for the activation of endothelial NF-kappaB, and C. pneumoniae may contribute to atherogenesis without active infection.

Previous studies have demonstrated that female reproductive hormones influence chlamydial infection both in vivo and in vitro. Due to the reduced availability of human genital tissues for research purposes, an alternative hormone-responsive model system was sought to study chlamydial pathogenesis. Mature female swine eliminated from breeding programs were selected as the animals of choice because of the similarity of a sexually transmitted disease syndrome and sequelae in swine to a disease syndrome and sequelae found in humans, because of the near identity of a natural infectious chlamydial isolate from swine to Chlamydia trachomatis serovar D from humans, and because a pig's epithelial cell physiology and the mean length of its estrous cycle are similar to those in humans. Epithelial cells from the cervix, uterus, and horns of the uterus were isolated, cultivated in vitro in Dulbecco's minimum essential medium-Hanks' F-12 (DMEM-F-12) medium with and without exogenous hormone supplementation, and analyzed for Chlamydia suis S-45 infectivity. The distribution of chlamydial inclusions in swine epithelial cells was uneven and was influenced by the genital tract site and hormone status. This study confirmed that, like primary human endometrial epithelial cells, estrogen-dominant swine epithelial cells are more susceptible to chlamydial infection than are progesterone-dominant cells. Further, the more differentiated luminal epithelial cells were more susceptible to infection than were glandular epithelial cells. Interestingly, chlamydial growth in mature luminal epithelia was morphologically more active than in glandular epithelia, where persistent chlamydial forms predominated. Attempts to reprogram epithelial cell physiology and thereby susceptibility to chlamydial infection by reverse-stage, exogenous hormonal supplementation were unsuccessful. Freshly isolated primary pig epithelial cells frozen at -80 degrees C in DMEM-F-12 medium with 10% dimethyl sulfoxide for several weeks can, after thawing, reform characteristic polarized monolayers in 3 to 5 days. Thus, primary swine genital epithelia cultured ex vivo appear to be an excellent cell model for dissecting the hormonal modulation of several aspects of chlamydial pathogenesis and infection.

Chlamydia trachomatis serovar E, the leading bacterial agent responsible for sexually transmitted diseases, is required to invade genital epithelial cells for its growth and survival, yet little is known about the adhesin-receptor interactions promoting its entry. In contrast, much has been published on the heparan sulfate receptor for binding C. trachomatis L2 elementary bodies (EBs) prior to entry into HeLa cells. Using a different experimental approach in which a biotinylated apical membrane protein receptor(s) attached to EB at 4 degrees C was stripped off the surface of polarized HEC-1B cells and immunoprecipitated with polyclonal anti-EB antibodies, an approximately 55-kDa protein was reproducibly detected by enhanced chemiluminescence and two-dimensional gel electrophoresis. Matrix-assisted laser desorption ionization mass-spectrometry sequence analysis revealed the 55-kDa protein to be protein disulfide isomerase (PDI), a member of the estrogen receptor complex which carries out thiol-disulfide exchange reactions at infected host cell surfaces. Exposure of HEC-1B cells during EB attachment (1.5 to 2 h) to three different inhibitors of PDI reductive reactions--(i) the thiol-alkylating reagent DTNB (5,5'-dithiobis[2-nitrobenzoic acid]), (ii) bacitracin, and (iii) anti-PDI antibodies--resulted in reduced chlamydial infectivity. Since (i) C. trachomatis serovar E attachment to estrogen-dominant primary human endometrial epithelial cells is dramatically enhanced and (ii) productive entry into and infectivity of EB in host cells is dependent on reduction of EB cross-linked outer membrane proteins at the host cell surface, these data provide some preliminary evidence for an intriguing new potential receptor candidate for further analysis of luminal C. trachomatis serovar E entry.

The inflammatory response associated with Chlamydia trachomatis genital infections is thought to be initiated by the release of proinflammatory cytokines from infected epithelial cells. This study focuses on the interactions between C. trachomatis-infected HeLa cells and immune cells involved in the early stages of infection, i.e., neutrophils and macrophages. First, we showed that the expression of interleukin-11 (IL-11), an anti-inflammatory cytokine mainly active on macrophages, was upregulated at the mRNA level in the genital tracts of infected mice. Second, incubation of differentiated THP-1 (dTHP-1) cells or monocyte-derived macrophages (MdM) with basal culture supernatants from C. trachomatis serovar E- or serovar L2-infected HeLa cells resulted in macrophage activation with a differential release of tumor necrosis factor alpha (TNF-alpha) and upregulation of indoleamine 2,3-deoxygenase (IDO) but not of Toll-like receptor 2 and 4 mRNA expression. Third, coculture of infected HeLa cells with dTHP-1 cells resulted in a reduction in chlamydial growth, which was more dramatic for serovar E than for L2 and which was partially reversed by the addition of anti-TNF-alpha antibodies for serovar E or exogenous tryptophan for both serovars but was not reversed by the addition of superoxide dismutase or anti-IL-8 or anti-IL-1beta antibodies. A gamma interferon-independent IDO mRNA upregulation was also detected in dTHP-1 cells from infected cocultures. Lastly, with a two-stage coculture system, we found that (i) supernatants from neutrophils added to the apical side of infected HeLa cell cultures were chlamydicidal and induced MdM to express antichlamydial activity and (ii) although polymorphonuclear leukocytes released more proinflammatory cytokines in response to serovar E- than in response to L2-infected cells, MdM were strongly activated by serovar L2 infection, indicating that the early inflammatory response generated with a nondisseminating or a disseminating strain is different.

Interleukin (IL)-10-deficient (IL-10-/-) mice develop colitis under specific pathogen-free (SPF) conditions and remain disease free if kept sterile (germ free [GF]). We used four different protocols that varied the time-points of oral administration of Lactobacillus plantarum 299v (L. plantarum) relative to colonization with SPF bacteria to determine whether L. plantarum could prevent and treat colitis induced by SPF bacteria in IL-10-/- mice and evaluated the effect of this probiotic organism on mucosal immune activation. Assessment of colitis included blinded histologic scores, measurements of secreted colonic immunoglobulin isotypes, IL-12 (p40 subunit), and interferon (IFN)-gamma production by anti-CD3-stimulated mesenteric lymph node cells. Treating SPF IL-10-/- mice with L. plantarum attenuated previously established colonic inflammation as manifested by decreased mucosal IL-12, IFN-gamma, and immunoglobulin G2a levels. Colonizing GF animals with L. plantarum and SPF flora simultaneously had no protective effects. Gnotobiotic IL-10-/- mice monoassociated with L. plantarum exhibited mild immune system activation but no colitis. Pretreatment of GF mice by colonization with L. plantarum, then exposure to SPF flora and continued probiotic therapy significantly decreased histologic colitis scores. These results demonstrate that L. plantarum can attenuate immune-mediated colitis and suggest a potential therapeutic role for this agent in clinical inflammatory bowel diseases.

Numerous investigations have shown that 70-kDa heat shock protein (Hsp70) homologs interact tightly with hydrophobic proteins and functionally assist proteins in membranous organelles and environments. One such protein is the Chlamydia trachomatis Hsp70 that is associated with isolated outer membrane complexes of infectious elementary bodies (EB). Previous observations have indicated that chlamydial Hsp70 plays a role in EB attachment to, or entry into, endometrial epithelial cells. In this study, immunofluorescence microscopy and transmission electron microscopy observations showed that chlamydial Hsp70 is not a surface-displayed ligand on purified EB. However, brief exposure of EB to the thiol reducing agent dithiothreitol (DTT) led to surface accessibility of the Hsp70 substrate-binding domain. Reduction of the highly disulfide-cross-linked EB outer membrane proteins with DTT resulted in a decrease in EB attachment and infectivity. Interestingly, exposure of EB to the membrane-impermeable thiol-alkylating reagent 5,5'-dithiobis(2-nitrobenzoic acid) enhanced attachment but compromised infectivity, suggesting that EB outer membrane proteins must be reduced for entry and productive infection. Together, our data suggest that (i) the structural integrity of the EB outer membrane, maintained by protein disulfide bonds, is important during the initial stages of attachment; (ii) reduction occurs within the localized microenvironment of host cell surfaces once intimate contact is established between EB and host cells; and (iii) subsequent conformational changes in EB ultrastructure allow productive infection in host cells. The accessibility of the Hsp70 substrate-binding domain may support the hypothesis that this protein plays a role in events following the initial stage of attachment instead of serving as a primary, surface-displayed adhesin.

Specific 3'-sulfogalactolipid [SGL-sulfogalactosyl ceramide (SGCer) and sulfogalactosylglycerolipid (SGG)] binding is compared for hsp70s cloned from Helicobacter pylori, Haemophilus influenzae, Chlamydia trachomatis serovar E, Escherichia coli, murine male germ cells, and the hsp70-like extracellular domain within the sperm receptor from Strongylocentrotus purpuratus. This lectin activity, conserved among the different hsp70 family members, is modulated by the SGL aglycone. This is shown by differential binding to both SGC fatty acid homologues and 3'-sulfogalactolipid neoglycoproteins generated by coupling bovine serum albumin (BSA) and glycosyl ceramide acids synthesized by oxidation of the double bond of sphingosine. Eukaryotic hsp70s preferentially bound the SGCer fatty acid homologues SG(24)Cer, SG(18)Cer, and SG(20:OH)Cer, while prokaryotic hsp70s bound SG(18:1)Cer and SG(20:OH)Cer. Eukaryotic hsp70s bound SGCer-BSA and SG(24)Cer-BSA conjugates where the latter is the main constituent in SGCer-BSA, while prokaryotic hsp70s bound SG(20:OH)Cer-BSA. None of the hsp70s bound sulfogalactosyl sphingosine (SGSph) or SGSph-BSA, further demonstrating the important role of the aglycone. Although the primary SGL recognition domain of all hsp70s is conserved, we propose that aglycone organization differentially influences the interaction with the sub-site. Heterogeneous SGCer aglycone isoforms in cells and the differential in vitro binding of eukaryotic and prokaryotic hsp70s may relate to their different adhesin roles in vivo as mediators of germ cell and bacterial/host interactions, respectively.

Chlamydiae are obligate intracellular bacterial pathogens whose entry into mucosal epithelial cells is required for intracellular survival and subsequent growth. After a seemingly stealthy entry, chlamydiae quickly modify their vacuole (i) for exit from the endosomal pathway to the exocytic pathway and (ii) to permit fusion with intercepted endoplasmic reticulum- and Golgi-derived vesicles carrying glycerophospholipids and sphingolipids for chlamydiae-containing vacuole membrane expansion. Chlamydiae possess novel hollow proteinaceous structures, termed projections, which they use to pierce the inclusion membrane, possibly to acquire from the epithelial cytoplasm nutrients they cannot synthesize; whether or not these truncated flagellar-like structures serve a dual exchange function for secretion of molecules to programme host cell signalling is unknown. Despite the accumulation of some 500-1000 progeny in the enormously enlarged inclusion, host cell function is surprisingly little disrupted, and progeny escape can be unobtrusive. This elegant adaptive pathogen strategy, which leads to silent, chronic human infection, is fascinating from a cellular microbiology perspective.

While genital infections caused by Chlamydia trachomatis are generally asymptomatic, the density and pattern of inflammation varies considerably. The purpose of this study was to try to dissect the signalling in chlamydiae-infected epithelial cells that triggers innate responses and regulates polymorphonuclear neutrophil (PMN) chemotaxis. Polarized endocervical epithelial HeLa cells, grown in commercial inserts, were inoculated either with the non-disseminating (luminal) serovar E or the disseminating serovar L2. At 12-48 h after infection, the chambers were used in a quantitative chemotaxis assay, and cytokine production by infected cells was examined using cDNA microarray technology and confirmed by enzyme-linked immunosorbent assay (ELISA). Infection of HeLa cells with C. trachomatis E or L2 induced a strong and similar PMN chemotactic response, but larger amounts of interleukin (IL)-8 and IL-11 were released after infection with serovar L2. IL-6 was also produced in modest amounts after infection with either strain, but no IL-1alpha or tumour necrosis factor (TNF)-alpha was detected in any of the culture supernatants tested. IL-11 did not appear to influence the PMN response to chlamydial infection, but secretion of large amounts of this anti-inflammatory cytokine, mainly active on macrophages, in the very early stages of the infection may allow C. trachomatis to escape some innate defences to establish infection.

An in vitro coculture model system was used to explore conditions that trigger neutrophil chemotaxis to Chlamydia trachomatis infected human epithelial cells (HEC-1B). Polarized HEC-1B monolayers growing on extracellular matrix (ECM) were infected with C. trachomatis serovar E. By 36 h, coincident with the secretion of chlamydial lipopolysaccharide and major outer membrane protein to the surfaces of infected cells, human polymorphonuclear neutrophils (PMNL) loaded with azithromycin migrated through the ECM and infiltrated the HEC-1B monolayer. Bioreactive azithromycin was delivered by the chemotactic PMNL to infected epithelial cells in concentrations sufficient to kill intracellular chlamydiae. However, residual chlamydial envelopes persisted for 4 weeks, and PMNL chemotaxis was triggered to epithelial cells containing residual envelopes. Infected endometrial cells demonstrated up-regulation of ENA-78 and GCP-2 chemokine mRNA. Thus, despite appropriate antimicrobial therapy, residual chlamydial envelope antigens may persist in infected tissues of culture-negative women and provide one source for sustained inflammation.

Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. Predominantly a cutaneous pathogen, H. ducreyi is present in chancroid ulcers that are characterized by extensive neutrophil accumulation in intraepidermal lesions accompanied by a mononuclear infiltrate in the dermis. We used an in vitro human skin model composed of foreskin fibroblasts and keratinocytes to examine host skin cell interactions with H. ducreyi 35000. Bacteria replicated and persisted in artificial skin for at least 14 days. We observed H. ducreyi inside suprabasal keratinocytes using transmission electron microscopy. Although no bacteria were seen in the basal keratinocyte region, these cells were disrupted in infected cocultures. H. ducreyi infection stimulated increased secretion of interleukin-6 (IL-6) and IL-8 by skin cells. Conversely, tumor necrosis factor alpha and IL-1alpha levels were not elevated. IL-8 produced in response to H. ducreyi infection may be involved in recruiting polymorphonuclear leukocytes and other inflammatory cells, thereby contributing to the tissue necrosis and ulcer formation characteristic of chancroid.

Unlike chlamydial lipopolysaccharide, which is released from the developing inclusion to the surface of infected genital epithelial cells, both Chlamydia trachomatis heat shock protein (hsp) 60 and 70 antigens remained confined within the inclusion during the course of the chlamydial developmental cycle. Exposure of the infected cells to penicillin to induce a persistent infection or to a lipophilic microbicide did not potentiate secretion or exocytosis of the chlamydial hsp.

Infection of eukaryotic cells by intracellular pathogens such as chlamydia requires attachment to the host cell surface. Chlamydia are thought to attach to the tips of microvilli in confluent monolayers of polarized cells. In vitro evidence obtained from migrating epithelial cells suggested that during healing the route of pathogen uptake might be different from that in intact epithelia. The small size of infectious chlamydial elementary bodies (approximately 0.3 microm in diameter) has made it difficult, however, to analyze the early stages of pathogen-host cell interaction in living cells by conventional microscopy. Contrast-enhanced video microscopy was therefore used to examine the earliest events of host-pathogen interaction and test the hypothesis that chlamydial uptake into the healing epithelia can involve translocation over the host cell surface. Observations made in this way were validated by scanning and immunofluorescence microscopy. These studies revealed two fates for chlamydiae taken onto the lamellipodial surface: 1) some chlamydiae were moved in a random fashion on the cell surface or were detached into the culture medium, whereas 2) other chlamydiae were translocated across the lamellipodium in a highly directed manner toward the microvillous perinuclear region. After internalization, these latter chlamydiae were found within intracellular inclusions, which demonstrated that this route of attachment and location of uptake resulted in productive growth.

Chlamydia trachomatis serovar E is one of the most common bacterial sexually transmitted pathogens. Since it is an obligate intracellular bacterium, efficient colonization of genital mucosal epithelial cells is crucial to the infectious process. Serovar E elementary bodies (EB) metabolically radiolabeled with 35S-Cys-Met and harvested from microcarrier bead cultures, which significantly improves the infectious EB-to-particle ratio, provided a more accurate picture of the parameters of attachment of EB to human endometrial epithelial cells (HEC-1B) than did less infectious 14C-EB harvested from flask cultures. Binding of serovar E EB was (i) equivalent at 35 and 4 degrees C, (ii) decreased by preexposure of EB to heat or the topical microbicide C31G, (iii) comparable among common eukaryotic cell lines (HeLa, McCoy), and (iv) significantly increased to the apical surfaces of polarized cells versus nonpolarized cells. In parallel experiments with C. trachomatis serovar L2, serovar E attachment was not affected by heparin or heparan sulfate whereas these glucosaminoglycans dramatically reduced serovar L2 attachment. These data were confirmed by competitive inhibition of serovar E binding and infectivity by excess unlabeled live and UV-inactivated serovar E EB but not by excess serovar L2 EB. The noninvasive serovar E strains in the lumen of the genital tract enter and exit the apical domains of target columnar epithelial cells to spread canalicularly in an ascending fashion from the lower to the upper genital tract. In contrast, the invasive serovar L2 strains are primarily submucosal pathogens and likely use the glucosaminoglycans concentrated in the extracellular matrix to colonize the basolateral domains of mucosal epithelia to perpetuate the infectious process.

Safe and effective vaginal microbicidal compounds are being sought to offer women an independent method for protection against transmission of sexually acquired pathogens. The purpose of this study was to examine the efficacy of two formulations of one such compound, C31G, against Chlamydia trachomatis serovar E alone, its host epithelial cell (HEC-1B) alone, and against chlamydiae-infected HEC-1B cells. Preexposure of isolated, purified infectious chlamydial elementary bodies (EB) to C31G, at pHs 7.2 and 5.7, for 1 h at 4 degrees C resulted in reduced infectivity of EB for HEC-1B cells. Examination of the C31G-exposed 35S-EB on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs and by Western blotting revealed a C31G concentration-dependent and pH-dependent destabilization of the chlamydial envelope, resulting in the release of chlamydial lipopolysaccharide and proteins. Interestingly, when the host human genital columnar epithelial cells were infected with chlamydiae and then exposed to dilute concentrations of C31G which did not alter epithelial cell viability, chlamydial infectivity was also markedly reduced. C31G gained access to the developing chlamydial inclusion causing damage to or destruction of metabolically active reticulate bodies as well as apparent alteration of the inclusion membrane, which resulted in premature escape of chlamydial antigen to the infected epithelial surface. These studies show that the broad-spectrum antiviral and antibacterial microbicide C31G also has antichlamydial activity.

Chlamydiae are obligate intracellular parasites which multiply within infected cells in a membrane-bound structure termed an inclusion. Newly internalized bacteria are surrounded by host plasma membrane; however, the source of membrane for the expansion of the inclusion is unknown. To determine if the membrane for the mature inclusion was derived by fusion with cellular organelles, we stained infected cells with fluorescent or electron-dense markers specific for organelles and examined inclusions for those markers. We observed no evidence for the presence of endoplasmic reticulum, Golgi, late endosomal, or lysosomal proteins in the inclusion. These data suggest that the expansion of the inclusion membrane, beginning 24 h postinoculation, does not occur by the addition of host proteins resulting from either de novo host synthesis or by fusion with preexisting membranes. To determine the source of the expanding inclusion membrane, antibodies were produced against isolated membranes from Chlamydia-infected mouse cells. The antibodies were demonstrated to be solely against Chlamydia-specified proteins by both immunoprecipitation of [35S]methionine-labeled extracts and Western blotting (immunoblotting). Techniques were used to semipermeabilize Chlamydia-infected cells without disrupting the permeability of the inclusion, allowing antibodies access to the outer surface of the inclusion membrane. Immunofluorescent staining demonstrated a ring-like fluorescence around inclusions in semipermeabilized cells, whereas Triton X-100-permeabilized cells showed staining throughout the inclusion. These studies demonstrate that the inclusion membrane is made up, in part, of Chlamydia-specified proteins and not of existing host membrane proteins.

Chlamydia trachomatis serovar L2 is an obligate intracellular bacterium which is internalized in target epithelial cells by endocytosis and resides within a membrane-bound vesicle. Over the next several hours following entry, individual serovar L2-containing vesicles fuse with one another to form a single membrane-bound vesicle (or inclusion) within which the microcolony develops. The experiments reported here directly examined the pH of vesicles containing chlamydiae. The pH was determined by measuring emission ratios of the fluorescent, pH-sensitive probe SNAFL (5-[and 6-]-carboxyseminaphthofluorescein-1, succinimidyl ester) conjugated to chlamydiae. The pH remained above 6.0 at 2, 4, and 12 h after infection, while the pH of vesicles contained heat-killed organisms fell 5.3. In the presence of amines, which raise the pH of acidic compartments, C. trachomatis inclusion formation was unaffected. Inactivation of Na+,K+ -ATPases, the ion pumps responsible for maintaining a pH above 6 within early endocytic vesicles, inhibited the growth of C. trachomatis within epithelial cells. Preventing vesicular acidification by inhibiting the vacuolar proton ATPase did not affect chlamydial growth. Thus, chlamydiae do not reside within highly acidic vesicles and avoid the pathway leading to lysosomes.

Objective: Our purpose was to study the effects of iron deprivation with and without human transferrin supplementation on the adherence and invasion of Neisseria gonorrhoeae to human endometrial cells., Study Design: N. gonorrhoeae grown with our without iron was placed in media alone or media containing 2.5 mg/ml saturated human transferrin or unsaturated transferrin. N. gonorrhoeae was inoculated onto polarized human endometrial carcinoma cell (HEC 1-B) monolayers, and at various intervals monolayers were washed and incubated with media containing gentamicin or media alone. Colony-forming units per milliliter of N. gonorrhoeae associated with HEC 1-B cells were then determined. N. gonorrhoeae strains tested included both a transferrin receptor-positive (wild-type) and a transferrin receptor-negative mutant. Differences in percent of original inoculum remaining at varying time points were analyzed by the Mann-Whitney U test. Transmission electron microscopy using a primary endometrial cell line was used to verify findings., Results: Iron-negative N. gonorrhoeae exhibited less adherence than did iron-positive N. gonorrhoeae. No difference in HEC 1-B adherence was seen when either saturated transferrin or unsaturated transferrin was added to the iron-positive N. gonorrhoeae. With iron-negative N. gonorrhoeae addition of either saturated transferrin or unsaturated transferrin significantly increased N. gonorrhoeae adherence although unsaturated transferrin did not permit growth of iron-negative N. gonorrhoeae in tissue culture media alone. Transmission electron microscopy confirmed increased adherence of iron-negative N. gonorrhoeae supplemented with unsaturated transferrin. An iron-negative N. gonorrhoeae mutant lacking the transferrin receptor exhibited no adherence regardless of addition of saturated transferrin or unsaturated transferrin. Invasion could not be quantitated reliably because of persistence of gentamicin effect., Conclusion: Iron and transferrin increased attachment of N. gonorrhoeae to human endometrial cells.

Chlamydia trachomatis serovar E is a major cause of bacterially-acquired sexually transmitted infections. Stock cultures of these obligate intracellular bacteria are often propogated in McCoy cells. We recently reported that greater infectious titers of chlamydiae could be obtained if the McCoy cells were cultured on collagen-coated microcarrier beads versus plastic flasks, although the reason for the difference in efficiency was not clear. This study analyzed the development of C. trachomatis grown in McCoy cells by the two methods. Transmission electron microscopy analysis revealed an accelerated chlamydial development, with maturation of reticulate bodies into elementary bodies sooner in McCoy cells grown on the porous substratum. Comparison of particle counts versus infectivity titers indicated the production of fewer numbers of elementary bodies but which were highly infectious sooner from the infected McCoy cell-microcarrier bead cultures than from duplicate infected McCoy cell cultures grown in plastic tissue culture flasks.

Genital serovariants of Chlamydia trachomatis establish infection by attachment, entry and multiplication within human endometrial epithelial cells. In previous studies, a chlamydial recombinant Escherichia coli was identified which exhibited a specific adherent phenotype to endometrial epithelial cells closely resembling that observed for a genital strain of C. trachomatis. One of the plasmid-encoded products expressed by the recombinant is a 28 kDa protein. In this study, localization of the 28 kDa protein in isolated outer membranes of recombinant E. coli and in chlamydial outer membrane complexes lends support for a potential role for this protein in the attachment process. Surprisingly, nucleic acid sequence analysis reveals that the 28 kDa protein shares a modest degree of homology with a member of the E. coli heat shock protein family.

Infection of genital epithelial cells by the closely related sexually transmitted pathogens Chlamydia trachomatis serovars E and L2 results in different clinical disease manifestations. Following entry into target host cells, individual vesicles containing chlamydiae fuse with one another to form one large inclusion. At the cellular level, the only obvious difference between these serovars is the time until inclusion maturation, which is 48 h for the invasive serovar L2 and 72 h for serovar E. To begin to define the intracellular events of these pathogens, the effect of cytoskeletal disruption on early endosome fusion and inclusion development in epithelial (HEC-1B) and fibroblast (McCoy) cells was analyzed by fluorescence microscopy. Disruption of microfilaments with cytochalasin D markedly reduced serovar E, but not serovar L2, infection of both cell lines. Conversely, microfilament as well as microtubule disruption, with colchicine or nocodazole, had no effect on serovar E inclusion development but resulted in the formation of multiple serovar L2 inclusions per cell during early and mid-development. Later in serovar L2 inclusion development (> 36 h postinfection), vesicles containing chlamydiae fused to form one large inclusion in the absence of an intact cytoskeleton. These results imply that (i) C. trachomatis serovar E may utilize a different pathway for uptake and development from serovar L2; (ii) these differences are consistent in both epithelial cells and fibroblasts; and (iii) the cytoskeleton plays a unique role in the infection of host cells by these two genital pathogens.

Infection of polarized human endometrial-gland epithelial cells obtained at hysterectomy with Chlamydia trachomatis serovar E may provide a relevant in vitro model for studies of pharmacokinetics in genital chlamydial infections. The minimal bactericidal concentration of azithromycin against C. trachomatis was lower in this model than in studies with nonpolarized cells (0.125 and 0.5 mg/L, respectively). Polarized cells also internalized more azithromycin over 24 hours. The results indicate that the eradication of chlamydial infections may be difficult to prove by antigen detection methods: the persistence of chlamydial envelope material within the intracellular vacuoles of azithromycin-treated cells may lead to a false-positive diagnosis of persisting chlamydial infection.

Chlamydia trachomatis is the leading cause of bacterially acquired sexually transmitted diseases in the United States and Europe. As an obligate intracellular pathogen, this bacterium must invade epithelial cells in order to survive and grow. Thus, multiple strategies probably exist for initial binding of chlamydiae to their target cells. Since a variety of bacteria have exploited integrins to colonize tissues, and a precedent existed for the involvement of extracellular matrix components in chlamydial attachment, this study first analyzed, by flow cytometry, integrins expressed by the human endometrial epithelial cell line HEC-1B. The genital cells were then exposed to monoclonal antibodies directed against those integrins and assayed for chlamydial attachment and inclusion development. Monoclonal antibodies bound to the alpha and/or beta 1 subunit of classic integrin receptors displayed by HEC-1B cells were not able to prevent colonization and infection of the epithelial cells by a genital isolate of C. trachomatis.

A polarized epithelial culture system and chlamydia-specific T-cell lines and clones were employed to investigate the ability and mechanisms by which T cells control the growth of chlamydiae in epithelial cells. Monolayers of polarized mouse epithelial cells were infected with the Chlamydia trachomatis agent of mouse pneumonitis (MoPn) and then exposed to antigen-stimulated MoPn-specific T-cell lines and clones. The results revealed that in vivo-protective MoPn-specific T-cell lines and clone 2.14-0 were capable of inhibiting the growth of MoPn in polarized epithelial cells. In contrast, the nonprotective MoPn-specific T-cell clone 2.14-3, naive splenic T cells, and a control T-cell clone could not inhibit the growth of MoPn in epithelial cells. Transmission electron microscopic analysis of infected epithelial cells which were exposed to clone 2.14-0 confirmed the absence of an established infection, as deduced from the virtual absence of inclusions in the cells. Antigen-specific activation of clone 2.14-0 was required for the MoPn-inhibitory function, since the absence of antigenic stimulation or stimulation with a heterologous chlamydial agent did not result in MoPn growth inhibition. Activation of clone 2.14-0 resulted in acquisition of the capacity to inhibit growth of both homologous (MoPn) and heterologous chlamydial agents. Close interaction between epithelial cells and clone 2.14-0 was required for the MoPn-inhibitory action, because separation of the cell types by a filter with a pore size of 0.45, 3.0, or even 8.0 microns abrogated MoPn inhibition. Protective T cells may act at close range in the epithelium to control chlamydial growth, possibly involving short-range-acting cytokines. The ability of antigen-stimulated T-cell lines and clones to inhibit chlamydial growth in polarized epithelial cultures could be a useful method for identifying protective T-cell clones and antigenic peptide fragments containing protective epitopes.

The 4.1-kb sequence of genomic DNA located upstream from the Chlamydia trachomatis grpE-like and dnaK-like heat shock (HS) genes was determined. Another putative HS gene was located just 5' to grpE along with an inverted repeat (IR) sequence proposed to be involved in HS regulation. The overall organization of this locus in Chlamydia resembles that of Bacillus subtilis, rather than Escherichia coli. Two other open reading frames (ORFs) were found in the sequence, one of which has homology to aminoacyl-tRNA synthetases. The other ORF has no significant homology to reported genes. We also examined the codon usage bias for these newly identified chlamydial ORFs and for previously reported chlamydial genes, and found them to be different from E. coli.

Electroporation was used to introduce DNA into the elementary bodies of the obligate parasitic bacterium Chlamydia trachomatis. The source of DNA for these experiments was the chimeric plasmid pPBW100, which was constructed from the well-characterized 7.5-kb plasmid of C. trachomatis and the Escherichia coli plasmid pBGS9. To select directly for C. trachomatis carrying pPBW100, an in-frame gene fusion between the chlamydial promoter P7248 and a promoterless chloramphenicol acetyltransferase (cat) cassette was incorporated into the plasmid. After infection of McCoy cells with electroporated elementary bodies containing pPBW100, the following were observed: (i) the plasmid DNA was detected inside the chloramphenicol-resistant chlamydial inclusions by in situ and Southern hybridization analyses; (ii) both physical and biochemical evidence showed that chloramphenicol acetyltransferase was synthesized by the electroporated C. trachomatis; (iii) expression of P7248::cat was developmentally regulated and occurred during the early stages of chlamydial reticulate body development; and (iv) although the expression from P7248::cat was mainly transient, there were rare instances where chloramphenicol-resistant C. trachomatis were observed after four passages.

Bacterial infections of the genital tract (gonorrhea, chlamydia, chancroid, syphilis) are common and cause significant morbidity. Their importance is heightened by recent appreciation of their roles in facilitation of transmission of the human immunodeficiency virus (HIV). Each is capable of causing repeated infections, suggesting lack of permanent broadly effective immunity. An effective vaccine has yet to be developed for any of these diseases. Rapid progress in understanding the molecular basis for pathogenesis of infection, including mechanisms for escape from otherwise effective immune surveillance and mechanisms for causing injury to host cells, has stimulated renewed efforts to make vaccines for some of these infections. Progress has been greatest for Neisseria gonorrhoeae and Chlamydia trachomatis. Present emphasis is on the major or principal outer membrane proteins of N. gonorrhoeae and C. trachomatis, based on evidence for neutralizing antibodies directed against surface-exposed variable domains of each of these proteins. Other surface-exposed proteins, including the iron-repressible transferrin receptor in gonococci and certain heat-shock proteins in chlamydia, also may be targets for vaccines. Although much remains to be learned, cautious optimism is warranted.

One route by which Chlamydia trachomatis is internalized into host endometrial epithelial cells is receptor-mediated endocytosis. Although this implies an adhesin-receptor interaction exists, specific chlamydial surface molecules have not been identified. We are investigating potential adhesin molecules using an in vitro functional assay to select for chlamydial recombinant Escherichia coli expressing an adherent phenotype. We have previously shown that E. coli JM109(pPBW58) attaches to epithelial cells by a specific process paralleling C. trachomatis and expresses at least three plasmid-encoded proteins (18, 28, and 82 kDa; Schmiel, D. H., Knight, S. T., Raulston, J. E., Choong, J., Davis, C. H., and Wyrick, P. B. (1991) Infect. Immun. 59, 4001-4012). In this report, we demonstrate that (i) the 82-kDa protein is associated with the outer membrane of both E. coli JM109-(pPBW58) and C. trachomatis serovar E elementary bodies; (ii) the plasmid-encoded protein is identical to the native chlamydial protein by mass, charge, antigenicity, and partial proteolytic peptide profiles; (iii) a highly homologous protein is present in C. trachomatis biovariant lymphogranuloma venereum; (iv) the 82-kDa protein is not covalently linked by disulfide bonds to other protein species in either E. coli JM109(pPBW58) or C. trachomatis; (v) sequence analysis of the open reading frame indicates this protein is a relative of the heat shock 70 family of proteins; and (vi) the inferred amino acid sequence contains a contiguous 73-amino acid region having 51% identity with the extracellular sperm receptor binding domain in Strongylocentrosus purpuratus (Foltz, K. R., Partin, J. S., and Lennarz, W. J. (1993) Science 259, 1421-1425). The potential involvement of an hsp70 protein in attachment may provide new insight on adherence mechanisms by obligate intracellular pathogens.