Diversity of Chlamydia trachomatis major outer membrane protein genes.

Genomic DNA libraries were constructed for Chlamydia trachomatis serovars B and C by using BamHI fragments, and recombinants that contained the major outer membrane protein (omp1) gene for each serovar were identified and sequenced. Comparisons between these gene sequences and the gene from serovar L2 demonstrated fewer base pair differences between serovars L2 and B than between L2 and C; this finding is consistent with the serologic and antigenic relationships among these serovars. The translated amino acid sequence for the major outer membrane proteins (MOMPs) contained the same number of amino acids for serovars L2 and B, whereas the serovar C MOMP contained three additional amino acids. The antigenic diversity of the chlamydial MOMP was reflected in four sequence-variable domains, and two of these domains were candidates for the putative type-specific antigenic determinant. The molecular basis of omp1 gene diversity among C. trachomatis serovars was observed to be clustered nucleotide substitutions for closely related serovars and insertions or deletions for distantly related serovars.     

Antigenic topology of chlamydial PorB protein and identification of targets for immune neutralization of infectivity

The outer membrane protein PorB is a conserved chlamydial protein that functions as a porin and is capable of eliciting neutralizing Abs. A topological antigenic map was developed using overlapping synthetic peptides representing the Chlamydia trachomatis PorB sequence and polyclonal immune sera. To identify which antigenic determinants were surface accessible, monospecific antisera were raised to the PorB peptides and were used in dot-blot and ELISA-based absorption studies with viable chlamydial elementary bodies (EBs). The ability of the surface-accessible antigenic determinants to direct neutralizing Ab responses was investigated using standardized in vitro neutralization assays. Four major antigenic clusters corresponding to Phe(34)-Leu(59) (B1-2 and B1-3), Asp(112) -Glu(145) (B2-3 and B2-4), Gly(179)-Ala(225) (B3-2 to B3-4), and Val(261)-Asn(305) (B4-4 to B5-2) were identified. Collectively, the EB absorption and dot-blot assays established that the immunoreactive PorB Ags were exposed on the surface of chlamydial EBs. Peptide-specific antisera raised to the surface-accessible Ags neutralized chlamydial infectivity and demonstrated cross-reactivity to synthetic peptides representing analogous C. pneumoniae PorB sequences. Furthermore, neutralization of chlamydial infectivity by C. trachomatis PorB antisera was inhibited by synthetic peptides representing the surface-exposed PorB antigenic determinants. These findings demonstrate that PorB Ags may be useful for development of chlamydial vaccines.     

Structural and functional analyses of the major outer membrane protein of Chlamydia trachomatis

Chlamydia trachomatis is a major pathogen throughout the world, and preventive measures have focused on the production of a vaccine using the major outer membrane protein (MOMP). Here, in elementary bodies and in preparations of the outer membrane, we identified native trimers of the MOMP. The trimers were stable under reducing conditions, although disulfide bonds appear to be present between the monomers of a trimer and between trimers. Cross-linking of the outer membrane complex demonstrated that the MOMP is most likely not in a close spatial relationship with the 60- and 12-kDa cysteine-rich proteins. Extraction of the MOMP from Chlamydia isolates under nondenaturing conditions yielded the trimeric conformation of this protein as shown by cross-linking and analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with different concentrations of acrylamide. Using circular dichroism spectroscopy, we determined that the trimers were formed mainly of beta-pleated sheet structures in detergent micelles. Using a liposomal swelling assay, the MOMP was found to have porin activity, and the size of the pore was estimated to be approximately 2 nm in diameter. The trimers were found to be stable in SDS at temperatures ranging from 4 to 37 degrees C and over a pH range of 5.0 to 8.0. In addition, the trimers of MOMP were found to be resistant to digestion with trypsin. In conclusion, these results show that the native conformation of the MOMP of C. trachomatis is a trimer with predominantly a beta-sheet structure and porin function.     

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.     

Induction of HLA class I-restricted CD8+ CTLs specific for the major outer membrane protein of Chlamydia trachomatis in human genital tract infections.

HLA class I-restricted CD8+ CTLs specific for the major outer membrane protein (MOMP) of Chlamydia trachomatis are present in the peripheral blood of humans who acquired genital tract infections with the organism. Three HLA-A2-restricted epitopes and two HLA-B51-restricted epitopes were identified in serovar E-MOMP. One of the five epitopes spans a variable segment of MOMP and is likely a serovar E-specific epitope. The other four epitopes are localized in constant segments and are C. trachomatis species specific. CTL populations specific for one or more of the four constant segment epitopes were isolated from all 10 infected subjects tested, regardless of infecting serovars, but from only one of seven uninfected subjects tested. The CTLs failed to recognize corresponding peptides derived from Chlamydia pneumoniae MOMP, further suggesting that they indeed resulted from genital tract infections with C. trachomatis. Significantly, ME180 human cervical epithelial cells productively infected with C. trachomatis were killed by the MOMP peptide-specific CTLs. Further investigations of the ability of such CTLs to lyse normal infected epithelial cells and their presence at inflamed sites in the genital tract will help understand the protective or pathological role of CTLs in chlamydial infections. The MOMP CTL epitopes may be explored as potential components of a subunit vaccine against sexually transmitted diseases caused by C. trachomatis. Moreover, the knowledge provided here will facilitate studies of HLA class I pathways of chlamydial Ag processing and presentation in physiologically relevant human APCs.     

Sequence analysis of the major outer membrane protein gene of an ovine abortion strain of Chlamydia psittaci

The major outer membrane protein (MOMP) gene from an ovine abortion strain of Chlamydia psittaci (S26/3) has been cloned and sequenced. The gene shows the features of other chlamydial MOMPs but comparison with the previously reported sequence for the ovine abortion isolate A22/M has revealed substantial sequence divergence which is clustered into the same four intramolecular regions as the sequence variation found between C. trachomatis serovars. Subsequent restriction enzyme analysis of A22/M DNA has shown that it has an avian-type genomic profile and thus the comparison is between types rather than between strains.     

Epitope mapping with solid-phase peptides: identification of type-, subspecies-, species- and genus-reactive antibody binding domains on the major outer membrane protein of Chlamydia trachomatis

The major outer membrane protein (MOMP) of Chlamydia trachomatis carries serovar-, subspecies-, species- and genus immunodomains, antibodies to which may be protective. We have compared the inferred amino acid sequences for MOMP from different serovars of C. trachomatis and from Chlamydia psittaci to identify the likely locations of these sero-taxonomic epitopes. Overlapping peptides corresponding to each of these regions were synthesized on a solid phase and probed with monoclonal antibodies (MAbs) of appropriate specificities. We describe the primary structures of the binding sites of MAb to each of these four epitopes on C. trachomatis serovar L1 MOMP.     

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.     

Overexpression and surface localization of the Chlamydia trachomatis major outer membrane protein in Escherichia coli

The Chlamydia trachomatis major outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP in Escherichia coli. The MOMP is surface exposed in C. trachomatis and capable of eliciting protective antibodies in infected hosts, and therefore has potential as a candidate vaccine to prevent infection with this significant human pathogen. The recombinant MOMP clone, L2rMOMP, contained the entire MOMP gene including the encoded leader sequence. Large quantities of chlamydial MOMP were expressed, some of which was processed and translocated to the E. coli surface. Surface localization of the MOMP was demonstrated by the binding of anti-MOMP monoclonal antibodies to the surface of the induced clone, and was visualized by fluorescence and electron microscopy. The induction of MOMP expression had a rapidly lethal effect on the L2rMOMP E. coli clone. Although no genetic system exists for Chlamydia, development of a stable, inducible E. coli clone which overexpresses the chlamydial MOMP permits a study of the biological properties of the MOMP, including the contribution of the MOMP variable segments to the topographical interactions which determine the antigenic structure responsible for human immune response.     

Sequence analysis and lipid modification of the cysteine-rich envelope proteins of Chlamydia psittaci 6BC.

The envelopes of elementary bodies of Chlamydia spp. consist largely of disulfide-cross-linked major outer membrane protein (MOMP) and two cysteine-rich proteins (CRPs). The MOMP gene of Chlamydia psittaci 6BC has been sequenced previously, and the genes encoding the small and large CRPs from this strain were cloned and sequenced in this study. The CRP genes were found to be tandemly arranged on the chlamydial chromosome but could be independently expressed in Escherichia coli. The deduced 87-amino-acid sequence of the small-CRP gene (envA) contains 15 cysteine residues, a potential signal peptide, and a potential signal peptidase II-lipid modification site. Hydropathy plot and conformation analysis of the small-CRP amino acid sequence indicated that the protein was unlikely to be associated with a membrane. However, the small CRP was specifically labeled in host cells incubated with [3H]palmitic acid and may therefore be associated with a membrane through a covalently attached lipid portion of the molecule. The deduced 557-amino-acid sequence of the large-CRP gene (envB) contains 37 cysteine residues and a single putative signal peptidase I cleavage site. In one recombinant clone the large CRP appeared to be posttranslationally cleaved at two sites, forming a doublet in a manner similar to the large-CRP doublet made in native C. psittaci 6BC. Comparison of the deduced amino acid sequences of the CRPs from chlamydial strains indicated that the small CRP is moderately conserved, with 54% identity between C. psittaci 6BC and Chlamydia trachomatis, and the large CRP is highly conserved, with 71% identity between C. psittaci and C. trachomatis and 85% identity between C. psittaci 6BC and Chlamydia pneumoniae. The positions of the cysteine residues in both CRPs are highly conserved in Chlamydia spp. From the number of cysteine residues in the MOMP and the CRPs and the relative incorporation of [35S]cysteine into these proteins, it was calculated that the molar ratio of C. psittaci 6BC elementary body envelope proteins is about one large-CRP molecule to two small-CRP molecules to five MOMP molecules.     

Characterization of Chlamydia trachomatis antigens with monoclonal and polyclonal antibodies

We used monoclonal antibodies (MAbs) to examine the antigenic specificity and biologic function of several Chlamydia trachomatis antigens. Thirteen distinct MAbs to eight C. trachomatis antigens were produced. Six MAbs reacted with unique epitopes on the major outer membrane protein (MOMP) and two of these had neutralizing activity. MAbs were produced to each of the chlamydial antigens with molecular masses of 10, 29, 32, 57, 60, 70, and 75 kilodaltons (kDa). These MAbs showed species and genus specificity in an immunoblot assay. None of the MAbs had neutralizing activity. The epitopes recognized on MOMP, 29-, and 10-kDa (presumably lipopolysaccharide) antigens were surface exposed. MAbs to the 75-kDa, 57-kDa, and MOMP antigens were used for immunoaffinity purification of these antigens to produce monospecific antisera in mice. With polyclonal sera, we found that the 75-kDa antigen was also immunoaccessible and that antibody to MOMP and 75-kDa antigens neutralized C. trachomatis infectivity. We conclude that, in addition to MOMP and lipopolysaccharide, antigens with molecular masses of 75 and 29 kDa are surface exposed. Antibodies to MOMP and 75-kDa antigens can neutralize the organism in vitro.     

Prediction of the membrane-spanning beta-strands of the major outer membrane protein of Chlamydia

There is preliminary experimental evidence indicating that the major outer-membrane protein (MOMP) of Chlamydia is a porin. We tested this hypothesis for the MOMP of the mouse pneumonitis serovar of Chlamydia trachomatis using two secondary structure prediction methods. First, an algorithm that calculates the mean hydrophobicity of one side of putative beta-strands predicted the positions of 16 transmembrane segments, a structure common to known porins. Second, outer loops typical of porins were assigned using an artificial neural network trained to predict the topology of bacterial outer-membrane proteins with a predominance of beta-strands. A topology model based on these results locates the four variable domains (VDs) of the MOMP on the outer loops and the five constant domains on beta-strands and the periplasmic turns. This model is consistent with genetic analysis and immunological and biochemical data that indicate the VDs are surface exposed. Furthermore, it shows significant homology with the consensus porin model of the program FORESST, which contrasts a proposed secondary structure against a data set of 349 proteins of known structure. Analysis of the MOMP of other chlamydial species corroborated our predicted model.     

Antigenic specificity of human antibody to chlamydia in trachoma and lymphogranuloma venereum

An understanding of the molecular basis of the humoral immune response to chlamydial infections in man requires the identification of target antigens to which antibodies are directed. The antigenic specificity of antibody from patients with lymphogranuloma venereum (LGV) or trachoma was therefore assessed by Western blotting. Surface polypeptides were first identified using purified chlamydial outer membrane complex as antigen. Antibodies in sera from patients with LGV but not from control negative sera reacted with a wide range of chlamydial surface polypeptides with molecular masses of 19, 29, 41, 58, 63 and 65 kDa. The major component of the antibody response detected by both immunoblotting and immunoprecipitation assay was directed against the major outer membrane protein (MOMP). Antibody to MOMP was species-specific on Western blotting, whereas antibody to several other polypeptides recognized common immunodeterminants on polypeptides of C. psittaci Cal-10 of equivalent molecular mass. Immunologically C. psittaci Cal-10 was more closely related to LGV strains of C. trachomatis than a guinea pig inclusion conjunctivitis strain of C. psittaci. Trachoma sera collected from a village in southern Iran showed predominantly type-specific antibody on micro-immunofluorescence to serotype A or B trachoma agents. These sera showed a weak immune response to MOMP, a pronounced response to a polypeptide of 36 kDa and much less widespread reactivity with other chlamydial polypeptides. The lack of an immune response to SDS-stable immunodeterminants on MOMP might contribute to the susceptibility of trachoma patients to repeated cycles of ocular infection with chlamydiae.     

High-level expression and epitope localization of the major outer membrane protein of Chlamydia trachomatis serovar L1

Fragments of the gene encoding the major outer membrane porin protein (MOMP) of Chlamydia trachomatis serovar L1 were ligated into the pUC plasmid vectors to give a series of overlapping recombinants expressing MOMP from the lac promoter. Induction of this promoter with IPTG leads to high-level expression of the recombinant porin protein. Electron microscopy shows the presence of insoluble inclusions within the Escherichia coli host cells. Probing the expressed MOMP fragments with a set of monoclonal antibodies permitted localization of the four binding sites (epitopes) of primary-sequence-dependent monoclonal antibodies that exhibit genus-, species-, subspecies- and type (serovar)-specific reactivities.     

Nucleotide sequence and taxonomic value of the major outer membrane protein gene of Chlamydia pneumoniae IOL-207

Chlamydia pneumoniae IOL-207 genomic DNA was hybridized with a 1.5 kb labelled DNA probe containing the 3' region of the coding sequence for the major outer membrane protein (MOMP) of C. trachomatis serovar L1. An 8.5 kb Bg/II fragment containing the complete MOMP gene was cloned into lambda EMBL3. Two hybridizing EcoRI fragments were sub-cloned into the lambda ZAP II cloning vector and the resulting plasmids were used as templates for sequencing both strands of the C. pneumoniae MOMP gene. Computer taxonomic studies using the nucleotide and inferred amino acid sequence of the MOMP of C. pneumoniae IOL-207 and all known chlamydial MOMP sequences supported the designation of C. pneumoniae as a new species, but electron microscope studies suggested that the presence of pear-shaped elementary bodies (EBs) may not be a reliable taxonomic criterion.     

Primary and secondary immune responses of mucosal and peripheral lymphocytes during Chlamydia trachomatis infection

Chlamydia trachomatis infection is followed by the development of antigen-specific cell-mediated immunity, which is detectable as a positive lymphocyte proliferation response to the chlamydial major outer membrane protein (MOMP) antigen. To date, however, there have been no studies on the mucosal immune responses to chlamydial antigens. This study aimed to study the primary and secondary immune responses of cervical lymphocytes in response to the chlamydial antigen. Median proliferative responses were found to be significantly (P<0.05) higher in patients with chlamydial infections than in controls. The chlamydial MOMP induced significantly higher IL-6 and IL-10 and lower interferon-gamma (IFN-gamma) secretion in cervical lymphocytes of Chlamydia-positive women, resulting in a T helper 2 response. On stimulation of peripheral blood mononuclear cells (PBMC) obtained from Chlamydia-positive women with the chlamydial antigen, the median levels of IL-10, IL-12 and IFN-gamma were higher than in controls, but the differences were not significant. Our study suggests that the mucosal immune responses towards Chlamydia trachomatis are different from those of PBMCs and are more helpful in understanding the cytokine responses in the female genital tract during chlamydial infection.     

Trafficking of chlamydial antigens to the endoplasmic reticulum of infected epithelial cells

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.     

Synthesis of disulfide-bonded outer membrane proteins during the developmental cycle of Chlamydia psittaci and Chlamydia trachomatis.

The disulfide bond cross-linked major outer membrane protein (MOMP) of the extracellular elementary bodies (EBs) of Chlamydia psittaci was reduced to its monomeric form within 1 h of entry of EBs into host cells by a process which was inhibited by chloramphenicol, while monomeric forms of three cross-linked cysteine-rich proteins could not be detected in Sarkosyl outer membrane complexes at any time in either extracellular or intracellular forms of C. psittaci. Synthesis and incorporation of the MOMP into outer membrane complexes were detected early in the infection cycle (12 h postinfection), while synthesis and incorporation of the cysteine-rich proteins were not observed until reticulate bodies had begun to reorganize into EBs at 20 to 22 h postinfection. By 46 h postinfection, the intracellular population of C. psittaci consisted mainly of EBs, the outer membrane complexes of which were replete with monomeric MOMP and cross-linked cysteine-rich proteins. Upon lysis of infected cells at 46 h, the MOMP was rapidly cross-linked, and infectious EBs were released. The status of the MOMP of intracellular Chlamydia trachomatis was similar to the status of the MOMP of C. psittaci in that the MOMP was largely uncross-linked at 24 and 48 h postinfection, but formed interpeptide disulfide bonds when it was exposed to an extracellular environment late in the developmental cycle. In contrast to C. psittaci, only a fraction of the cross-linked MOMP of infecting EBs of C. trachomatis was reduced by 4 h postinfection, and reduction of the MOMP was not inhibited by chloramphenicol. Exposure of extracellular EBs of C. trachomatis and C. psittaci to dithiothreitol reduced the MOMP but failed to stimulate metabolic activities normally associated with reticulate bodies.     

Identification of immunodominant linear B-cell epitopes within the major outer membrane protein of Chlamydia trachomatis

Chlamydia trachomatis is one of the most prevalent sexually transmitted pathogens. Chlamydial major outer membrane protein (MOMP) can induce strong cellular and humoral immune responses in murine models and has been regarded as a potential vaccine candidate. In this report, the amino acid sequence of MOMP was analyzed using computer-assisted techniques to scan B-cell epitopes, and three possible linear B-cell epitopes peptides (VLKTDVNKE, TKDASIDYHE, TRLIDERAAH) with high predicted antigenicity and high conservation were investigated. The DNA coding region for each potential epitope was cloned into pET32a(+) and expressed as Trx-His-tag fusion proteins in Escherichia coli. The fusion proteins were purified by Ni-NTA agarose beads and followed by SDS-PAGE and western blot analysis. We immunized mice with these three fusion proteins. The sera containing anti-epitope antibodies from the immunized mice could recognize C. trachomatis serovars D and E in ELISA. Antisera of these fusion proteins displayed an inhibitory effect on invasion of serovar E by in vitro neutralization assays. In addition, serum samples from convalescent C. trachomatis-infected patients were reactive with the epitope fusion proteins by western blot assay. Our results showed that the epitope sequences selected by bioinformatic analysis are highly conserved C. trachomatis MOMP B-cell epitopes, and could be good candidates for the development of subunit vaccines, which can be used in clinical diagnosis.