The role of lipopolysaccharide in the exposure of protective antigenic sites on the major outer membrane protein of Chlamydia trachomatis.

A species-specific monoclonal IgM antibody (mAb) 9BF8 directed against the major outer membrane protein (MOMP) of Chlamydia trachomatis neutralized several chlamydial serovars in a complement-independent manner. The presence of Mg2+ ions negated the neutralization in serovars F, L1 and L2, but not in serovars A, B, E, D and K. The ability of monovalent Fab-fragments of this mAb to neutralize chlamydial infectivity in a Mg-independent manner suggested that conformational alterations on the chlamydial surface induced by the cation hindered the IgM but allowed the smaller Fab fragment access to its epitope. In order to determine the chlamydial component that binds Mg, elementary bodies (EB) of serovars E and L1 were treated with EDTA at pHs 8 and 9. The infectivity of the treated EB and the amount of released LPS were determined. Only after EDTA treatment at pH 9, as the LPS release increased, did the binding of the mAb on the chlamydial surface become Mg-independent. The infectivity of the EB was almost completely lost after such a treatment. These results suggest that the chlamydial LPS has the potential to modulate the exposure of antigenic sites on the MOMP, when it is cross-linked by Mg2+. They further imply that serovars protected by Mg and those that are not differ in the surface topology of one particular MOMP epitope, but are antigenically very similar. This difference might be of considerable importance in vivo.     

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.     

Epithelial membrane protein 2 modulates infectivity of Chlamydia muridarum (MoPn)

Chlamydiae are bacterial pathogens which have evolved efficient strategies to enter, replicate, and survive inside host epithelial cells, resulting in acute and chronic diseases in humans and other animals. Several candidate molecules in the host receptor complex have been identified, but the precise mechanisms of infection have not been elucidated. Epithelial membrane protein-2 (EMP2), a 4-transmembrane protein, is highly expressed in epithelial cells in sites of chlamydial infections. Here we show that infectivity of the Chlamydia muridarum (MoPn) is associated with host cellular expression of EMP2 in multiple cell lines. Recombinant knockdown of EMP2 impairs infectivity, whereas infectivity is augmented in cells recombinantly modified to over-express EMP2. An epithelial cell line without native expression of EMP2 is relatively resistant to MoPn infection, whereas infectivity is markedly increased by recombinant expression of EMP2 in that cell line. Blockade of surface EMP2 using a specific anti-EMP2 antibody significantly reduces chlamydial infection efficiency. In addition, MoPn infectivity as measured in the EMP2 overexpressing cell line is not heparin-dependent, suggesting a possible role for EMP2 in the non-reversible phase of early infection. These findings identify EMP2 as a candidate host protein involved in infection of C. muridarum (MoPn).     

Transmission of Chlamydia pneumoniae infection from blood monocytes to vascular cells in a novel transendothelial migration model

Chlamydia pneumoniae uses blood monocytes (PBMC) for systemic dissemination, persists in atherosclerotic lesions, and has been implicated in the pathogenesis of atherosclerosis. During transmigration in a newly developed transendothelial migration model (TEM) C. pneumoniae-infected PBMC spread their infection to endothelial cells. Transmigrated PBMC retained their infectivity and transmitted the pathogen to smooth muscle cells in the lower chamber of the TEM. Detection of chlamydial HSP60 mRNA proved pathogen viability and virulence. We conclude that PBMC can spread chlamydial infection to vascular wall cells and we suggest the TEM as a novel tool to analyze host-pathogen interactions in vascular chlamydial infections.     

Blockade of epithelial membrane protein 2 (EMP2) abrogates infection of Chlamydia muridarum murine genital infection model

New methods are needed to eradicate or prevent Chlamydia trachomatis infections. Blockade of epithelial membrane protein 2 (EMP2) by genetic silencing or neutralizing polyclonal antibody reduced chlamydial infectivity in vitro . This study tests the prediction that recombinant anti-EMP2 diabody could reduce early chlamydial infection of the genital tract in vivo . In a murine infection model, pretreatment with anti-EMP2 diabody, as compared with control diabody, significantly reduced bacterial load, tissue production of inflammatory cytokines, recruitment of polymorphonuclear leukocytes, and local tissue inflammation. These findings support EMP2 as a potential preventative and therapeutic target for genital chlamydial infection.     

Control mechanisms governing the infectivity of Chlamydia trachomatis for HeLa cells: the role of calmodulin

Adhesion of the obligate intracellular bacterium Chlamydia trachomatis to host cells is associated with a flux of Ca2+ across the cell membrane, and infection is enhanced by treatment of host cells with Ca2+ ionophore. The possibility that Ca2+ might interact with host cell Ca2+ regulatory proteins to promote chlamydial infection was investigated. Treatment of HeLa 229 cells with the calmodulin inhibitors pimozide, trifluoperazine, chlorpromazine, promethazine or haloperidol reduced chlamydial infectivity as measured by inclusion counting or the specific incorporation of [3H]threonine. The inhibitory effect was reversible, dose-related and shown to be associated with impairment of chlamydial adhesion and uptake by the host cells. This effect was clearly distinguished from the delayed maturation of chlamydiae due to continuous exposure to calmodulin inhibitors which may result from a decrease in the availability of high energy compounds from the host cells necessary for chlamydial growth. The possible mechanisms for calmodulin-mediated chlamydial endocytosis are discussed.     

Purification on Renografin Density Gradients of Chlamydia trachomatis Grown in the Yolk Sac of Eggs

Chlamydia trachomatis grown in the yolk sac of embryonated eggs was purified by centrifugation on continuous isopycnic Renografin density gradients. A band of chlamydial particles with a buoyant density of 1.20 contained 70% of the starting particles, and electron microscopy revealed the virtual absence of contaminating egg material. Centrifugation on Renografin gradients caused only a moderate decrease in infectivity. For large-scale purification, infected yolk sac was centrifuged through Renografin solutions, resulting in greater than 60% recovery of starting chlamydial particles, but less than 1% recovery of the dry weight and protein.     

Trachoma and LGV biovars of Chlamydia trachomatis share the same glycosaminoglycan-dependent mechanism for infection of eukaryotic cells

A sulphated glycosaminoglycan-dependent mechanism of microbial infection for mammailan cells was characterized for the Chlamydia trachomatis trachoma and lymphogranuloma venereum (LGV) biovars. We demonstrated that the trachoma and LGV biovars compete for the same receptor(s) on host cells and that their infectivity was inhibited by heparin or heparan sulphate. Using a specific heparan suiphate lyase (heparitinase) to treat organisms, the Infectivity of both biovars was abolished. Furthermore, exogenous heparan sulphate rescued chlamydial infectivity following treatment with heparitinase and the restored infectivity was neutralized by an anti-heparan sulphate monoclonal antibody. These data suggest that heparan sulphate-like-mediated Interactions between C. trachomatis and eukaryotic cells are essential for infectivity.     

Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis

A panel of monoclonal antibodies (MAb) was generated against Chlamydia trachomatis serovar B, an etiologic agent of blinding trachoma. The specificities of MAb were determined by dot blot assay by using viable elementary bodies of 13 C. trachomatis serovars and two C. psittaci strains. The dot blot assay was used to identify those antigens that were unique and immunoaccessible on the chlamydial surface. MAb were identified that recognized bi-specific (serovars B and Ba) or subspecies-specific (various B complex serovars) surface-exposed antigenic determinants that were either resistant or sensitive to heat denaturation (56 degrees C, 30 min). All of the MAb recognized the major outer membrane protein as determined by either immunoblotting or radioimmunoprecipitation. MAb specific for immunoaccessible major outer membrane protein epitopes protected mice from toxic death after i.v. injection of B serovar elementary bodies and neutralized the infectivity of the organism for monkey eyes. In contrast, MAb reactive against non-immunoaccessible subspecies- or species-specific major outer membrane protein epitopes or against an immunoaccessible genus-specific epitope located on chlamydial lipopolysaccharide did not protect mice from toxic death or neutralize infectivity of the parasite for monkey eyes. These data suggest that those major outer membrane protein antigenic determinants that are serovar or serogroup specific and are accessible to antibody on the chlamydial cell surface may be useful as a recombinant subunit vaccine for trachoma.     

Localization and characterization of a putative cysteine desulfurase in Chlamydia psittaci

Chlamydia psittaci is an obligate intracellular pathogen with a biphasic developmental life cycle. It is auxotrophic for a variety of essential metabolites and obtains amino acids from eukaryotic host cells. Chlamydia can develop inside host cells within chlamydial inclusions. A pathway secreting proteins from inclusions into the host cellular cytoplasm is the type III secretion system (T3SS). The T3SS is universal among several Gram-negative bacteria. Here, we show that CPSIT_0959 of C. psittaci is expressed midcycle and secreted into the infected cellular cytoplasm via the T3SS. Recombinant CPSIT_0959 possesses cysteine desulfurase and PLP-binding activity, which removes sulfur from cysteine to produce alanine, and helps chlamydial replication. Our study shows that CPSIT_0959 improve the infectivity of offspring elementary bodies and seems to promote the replication by its product. This phenomenon has inhibited by the PLP-dependent enzymes inhibitor. Moreover, CPSIT_0959 increased expression of Bim and tBid, and decreased the mitochondrial membrane potential of host mitochondria to induce apoptosis in the latecycle for release of offspring. These results demonstrate that CPSIT_0959 has cysteine desulfurase and PLP-binding activity and is likely to contribute to apoptosis of the infected cells via a mitochondria-mediated pathway to improve the infectivity of progeny.     

Inhibitory effect of heparan sulfate-like glycosaminoglycans on the infectivity of Chlamydia pneumoniae in HL cells varies between strains

Glycosaminoglycans are known to participate in the attachment of several chlamydial strains. We studied the effect of heparin, enoxaparin, low-molecular-weight heparin, chondroitin sulfate A, and heparinase I on the infectivity of Chlamydia pneumoniae strain CWL029 and two Finnish isolates, Kajaani 7 and Parola, in an HL cell line which is epithelial in origin. Two Chlamydia trachomatis strains, L2 and E, were used for comparison. The infectivity of all C. pneumoniae strains and C. trachomatis serovar E was inhibited not only by heparin derivatives but also by chondroitin sulfate A and heparinase treatment. Treatment of host cells with heparin derivatives and heparinase was also inhibitory. Different chlamydial strains and species seem, however, to vary in their ability to use heparin in their attachment to host cells.     

An early event in the herpes simplex virus type-2 replication cycle is sufficient to induce Chlamydia trachomatis persistence

Epidemiological studies have demonstrated that co-infections of herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occur in vivo. Data from a tissue culture model of C. trachomatis/HSV-2 co-infection indicate that viral co-infection stimulates the formation of persistent chlamydiae. Transmission electron microscopic (TEM) analyses demonstrated that in both HeLa and HEC-1B cells, co-infection caused developing chlamydiae to exhibit swollen, aberrantly shaped reticulate bodies (RBs), characteristically observed in persistence. Additionally, HSV-2 co-infection suppressed production of infectious chlamydial elementary bodies (EBs) in both host cell types. Co-infection with HSV type 1 (HSV-1) produced similar morphologic alterations and abrogated infectious EB production. These data indicate that virus-induced chlamydial persistence was neither host cell- nor virus strain-specific. Purification of crude HSV-2 stocks demonstrated that viral particles were required for coinfection-induced chlamydial persistence to occur. Finally, co-infection with either UV-inactivated, replication-incompetent virus or replication-competent HSV-2 in the presence of cyclohexamide reduced chlamydial infectivity without altering chlamydial genomic DNA accumulation. These data demonstrate that productive viral replication is not required for the induction of chlamydial persistence and suggest that HSV attachment and entry can provide the necessary stimulus to alter C. trachomatis development.     

The developmental cycle of Chlamydia trachomatis in McCoy cells treated with cytochalasin B.

The growth of a genital trachoma-inclusion conjunctivitis agent strain of Chlamydia trachomatis in McCoy cells treated with cytochalasin B was studied by quantitative infectivity estimations and by light and electron microscopy. Provided that infection of the monolayer was initiated by centrifuging the infectious particles on to the cells before incubation, this chlamydial strain grew as fast and to as high a titre [approximately 10(7) inclusion-forming units (i.f.u.) per culture] as those chlamydiae which infect cell cultures in vitro without centrifugation. Each i.f.u. inoculated yielded approximately 600 i.f.u., and extracellular infectivity was detected soon after intracellular infectivity appeared. Inclusions were recognized by fluorescent antibody staining techniques early in the developmental cycle when cultures were not infectious and when only reticulate bodies were seen by electron microscopy. Inclusions were recognized in Giemsa-stained preparations examined by dark ground microscopy only when elementary bodies appeared in the inclusions. Iodine staining was not a reliable indicator either of the number of inclusions present or of their infectivity.     

Chlamydia attachment to mammalian cells requires protein disulfide isomerase

For Chlamydia, an intracellular pathogen of humans, host cell invasion is obligatory for survival, growth and pathogenesis. At the molecular level, little is known about the binding and entry of Chlamydia into the mammalian host cell. Chlamydia are genetically intractable therefore experimental approaches targeting the host are often necessary. CHO6 is a mutagenized cell line resistant to attachment and infection by Chlamydia. In this study, CHO6 was shown using proteomic methods to have a defect in processing of the leader sequence for protein disulfide isomerase (PDI). Complementation by expression of full-length PDI restored C. trachomatis binding and infectivity in the CHO6 mutant cell line. The cell line was also resistant to diphtheria toxin and required complemented cell-surface PDI for toxin entry. These data demonstrate that native PDI at the cell surface is required for effective chlamydial attachment and infectivity.     

Reversibility of heat shock in Chlamydia trachomatis

The heat shock effect on chlamydia development was studied. We report here that the reversibility of the heat shock response did not depend on the stage of chlamydial morphogenesis at which transfer to high temperature occurred, and the infectivity of the particles produced was not affected significantly, so long as the heat shock exposure was not prolonged. Exposure to heat shock for more than 9 h resulted in stagnation of the growth cycle, appearance of aberrant reticulate body particles and loss of infectivity. SDS-PAGE analysis of proteins synthesized under prolonged heat shock showed increased relative abundance of heat shock proteins in common with other procaryotic organisms.     

Autophagy restricts Chlamydia trachomatis growth in human macrophages via IFNG-inducible guanylate binding proteins

Interferon γ (IFNG) is a key host response regulator of intracellular pathogen replication, including that of Chlamydia spp The antichlamydial functions of IFNG manifest in a strictly host, cell-type and chlamydial strain dependent manner. It has been recently shown that the IFNG-inducible family of immunity-related GTPases (IRG) proteins plays a key role in the defense against nonhost adapted chlamydia strains in murine epithelial cells. In humans, IFN-inducible guanylate binding proteins (hGBPs) have been shown to potentiate the antichlamydial effect of IFNG; however, how hGBPs regulate this property of IFNG is unknown. In this study, we identified hGBP1/2 as important resistance factors against C. trachomatis infection in IFNG-stimulated human macrophages. Exogenous IFNG reduced chlamydial infectivity by 50 percent in wild-type cells, whereas shRNA hGBP1/2 knockdown macrophages fully supported chlamydial growth in the presence of exogenous IFNG. hGBP1/2 were recruited to bacterial inclusions in human macrophages upon stimulation with IFNG, which triggered rerouting of the typically nonfusogenic bacterial inclusions for lysosomal degradation. Inhibition of lysosomal activity and autophagy impaired the IFNG-mediated elimination of inclusions. Thus, hGBP1/2 are critical effectors of antichlamydial IFNG responses in human macrophages. Through their capacity to remodel classically nonfusogenic chlamydial inclusions and stimulate fusion with autophagosomes, hGBP1/2 disable a major chlamydial virulence mechanism and contribute to IFNG-mediated pathogen clearance.     

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.     

Damage/Danger Associated Molecular Patterns (DAMPs) Modulate Chlamydia pecorum and C. trachomatis Serovar E Inclusion Development In Vitro

Persistence, more recently termed the chlamydial stress response, is a viable but non-infectious state constituting a divergence from the characteristic chlamydial biphasic developmental cycle. Damage/danger associated molecular patterns (DAMPs) are normal intracellular components or metabolites that, when released from cells, signal cellular damage/lysis. Purine metabolite DAMPs, including extracellular ATP and adenosine, inhibit chlamydial development in a species-specific manner. Viral co-infection has been shown to reversibly abrogate Chlamydia inclusion development, suggesting persistence/chlamydial stress. Because viral infection can cause host cell DAMP release, we hypothesized DAMPs may influence chlamydial development. Therefore, we examined the effect of extracellular ATP, adenosine, and cyclic AMP exposure, at 0 and 14 hours post infection, on C. pecorum and C. trachomatis serovar E development. In the absence of de novo host protein synthesis, exposure to DAMPs immediately post or at 14 hours post infection reduced inclusion size; however, the effect was less robust upon 14 hours post infection exposure. Additionally, upon exposure to DAMPs immediately post infection, bacteria per inclusion and subsequent infectivity were reduced in both Chlamydia species. These effects were reversible, and C. pecorum exhibited more pronounced recovery from DAMP exposure. Aberrant bodies, typical in virus-induced chlamydial persistence, were absent upon DAMP exposure. In the presence of de novo host protein synthesis, exposure to DAMPs immediately post infection reduced inclusion size, but only variably modulated chlamydial infectivity. Because chlamydial infection and other infections may increase local DAMP concentrations, DAMPs may influence Chlamydia infection in vivo, particularly in the context of poly-microbial infections.     

The 32-kDa glycoprotein of Chlamydia trachomatis is an acidic protein that may be involved in the attachment process

Abstract The 32-kDa glycoprotein of Chlamydia trachomatis was shown to have a p I of 6.2 to 6.4 which distinguished this protein from the chlamydial histone-like protein of similar molecular mass that has a p I of > 10. The initial interaction of the glycan of 32 kDa glycoprotein and HeLa cells was also investigated. Glycan was cleaved from the protein backbone by N -glycanase and radiolabeled with tritium by sodium borohydride reduction. Competition assays showed the binding of glycan to HeLa cells was inhibited by galactose, mannose, and N -acetylglucosamine but not by sedoheptulose and fructose. Untreated and UV-treated organisms inhibited the binding, while heat-inactivated organisms did not. Binding was blocked by rabbit antiserum against whole organisms but not by rabbit anti-155-kDa antiserum or monoclonal antibodies against the lipopolysaccharide and major outer membrane protein.