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At least 19 recordsLinked to original sources

PubMed1990

Complement component C3 fixes selectively to the major outer membrane protein (MOMP) of Legionella pneumophila and mediates phagocytosis of liposome-MOMP complexes by human monocytes.

Legionella pneumophila is a facultative intracellular bacterial pathogen that parasitizes human monocytes and alveolar macrophages. Previous studies from this laboratory have shown that monocyte complement receptors CR1 and CR3 and complement component C3 in serum mediate L. pneumophila phagocytosis. In this study, we have explored C3 fixation to L. pneumophila. We developed a whole-cell enzyme-linked immunosorbent assay (ELISA) to measure C3 fixation to the bacterial surface. By this assay, C3 fixes to L. pneumophila that are opsonized in fresh nonimmune serum, and C3 fixation takes place via the alternative pathway of complement activation. Immunoblot analysis of opsonized L. pneumophila indicated that C3 fixes selectively to specific acceptor molecules of L. pneumophila. Consistent with this, when nitrocellulose blots of whole L. pneumophila or bacterial components are incubated in fresh nonimmune serum, C3 fixes exclusively to the major outer membrane protein (MOMP) of L. pneumophila, a porin; C3 does not fix to L. pneumophila LPS on these blots. To further explore the role of MOMP in C3 fixation and phagocytosis, we reconstituted purified MOMP into liposomes. By the ELISA, MOMP-liposomes, but not plain liposomes lacking MOMP, avidly fix C3. Consistent with a dominant role for MOMP in C3 fixation, MOMP-liposomes form a C3 complex of the same apparent molecular weight as whole L. pneumophila in nonimmune serum. Opsonized radioiodinated MOMP-liposomes avidly adhere to monocytes, and adherence is dose dependent upon serum. By electron microscopy, opsonized MOMP-liposomes are efficiently phagocytized by human monocytes, and phagocytosis takes place by a conventional appearing form of phagocytosis. This study demonstrates that C3 fixes selectively to the MOMP of L. pneumophila, and that, in the presence of nonimmune serum, MOMP can mediate phagocytosis of liposomes and, potentially, phagocytosis of intact L. pneumophila by human monocytes.

Animals↗

PubMed1990

Antibody response to epitopes of chlamydial major outer membrane proteins on infectious elementary bodies and of the reduced polyacrylamide gel electrophoresis-separated form.

Approximately 60% of the outer membrane of chlamydial elementary bodies (EBs) consists of the major outer membrane protein (MOMP) that has structural and metabolic functions. The antigenic properties of MOMPs from mammalian strains of serovars 1 and 2 and an avian strain of Chlamydia psittaci were analyzed. Polyclonal-monospecific antisera (PMAs), one monoclonal antibody (MAb), and polyclonal antisera (PAs) were produced against reduced polyacrylamide gel electrophoresis-separated MOMPs and against infectious EBs. Three PMAs and the MAb, which were induced by reduced polyacrylamide gel electrophoresis-separated MOMPs, reacted strongly in Western blot (immunoblot) assays with MOMPs of serovar 1 and 2 strains as well as with that of the avian strain 6BC, and two of these PMAs reacted weakly (dilution, 1:20) with the MOMP of strain LGV-2. The third PMA and the MAb against the MOMP of the serovar 2 strain did not react with the MOMP of LGV-2. Four PAs were produced against infectious EBs of the serovar 1 strain. One of these PAs reacted with the homologous MOMP and that of the avian strain 6BC but did not recognize MOMPs of other chlamydial strains. Three of the PAs reacted with MOMPs of homologous strains only and failed to recognize MOMPs of avian, serovar 2, and LGV-2 strains. Five PAs induced against infectious EBs of the serovar strain 2 reacted only with the MOMPs of the homologous strains and failed to recognize MOMPs of other strains of chlamydiae. Consequently, MOMPs of C. psittaci strains possess genus-, species-, and serovar-specific epitopes whereby the immune response to serovar-specific epitopes of MOMP predominate when infectious EBs are used for immunization.

Animals↗

PubMed1992

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.

Antibodies, Bacterial↗

PubMed1985

Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin.

We have purified the major outer membrane protein (MOMP) of Legionella pneumophila, determined that it is associated with peptidoglycan, and characterized it as a porin. To purify the MOMP, we used a simple, rapid, three-step procedure that gave us the protein in high yield. The first step of the purification procedure involved selectively extracting the MOMP from whole bacterial cells with calcium and zwitterionic detergent. The second and third steps achieved purification by ion-exchange and molecular-sieve chromatography. The dissociation of the MOMP into monomers was dependent upon the presence of a reducing agent and was enhanced by treatment at 100 degrees C. To study the relationship of the MOMP to peptidoglycan, we extracted the protein by a modification of the Rosenbusch procedure. Like the Escherichia coli porins, the MOMP was peptidoglycan associated. The MOMP was at least partially dissociated from peptidoglycan in sodium dodecyl sulfate and a high salt concentration. To study the ion channel-forming properties of the MOMP, we reconstituted the MOMP in planar lipid membranes. The MOMP formed ion-permeable channels with a single-channel conductance size of 100 picoSiemens. The MOMP channels exhibited a fourfold selectivity for cations over anions and voltage-independent gating. These findings demonstrate that the MOMP is a porin with properties similar to those of E. coli porins.

Bacterial Outer Membrane Proteins↗

PubMed1990

Identification and characterization of T helper cell epitopes of the major outer membrane protein of Chlamydia trachomatis.

Chlamydia trachomatis serovars A, B, and C are the causative agents of trachoma, the world's leading cause of preventable blindness. Immunoprophylaxis is a possible approach to control trachoma. The chlamydial major outer membrane protein (MOMP) is thought to play an important role in the development of protective immunity against chlamydial infection, and is therefore considered to be a promising candidate antigen in the development of a trachoma vaccine. Much effort has been focused on the molecular characterization of B cell sites of the MOMP that elicit neutralizing antibodies. Neutralizing sites have been identified as linear epitopes that reside within variable domains (VDs) of the protein whose primary sequences vary among different serovars. No information exists on MOMP T helper (Th) cell antigenic determinants, which are likely critical components for the development of a successful chlamydial vaccine. We used overlapping synthetic peptides (25 mers) representing the entire primary sequence of serovar A MOMP in T cell proliferation assays to identify T cell antigenic determinants of this molecule. Eight synthetic peptides (A-2, A-3, A-7, A-8, A-11, A-22, A-23, and A-24) stimulated proliferative responses of splenic T cells isolated from MOMP-immunized A/J mice. To ascertain if these peptides functioned as Th cell antigens, we determined their ability to prime A/J mice in vivo to produce an anamnestic IgG response specific to the MOMP. Mice primed with synthetic peptides A-8 (106-130) or A-23 (331-355) produced IgG antibodies reactive with the native MOMP and with the synthetic peptides corresponding to surface-accessible serovar-specific epitopes located in VD I and serogroup-specific epitopes located in VD IV of the protein. We synthesized the A-8 and A-23 peptides with the VD I sequence as colinear chimeric peptides. Immunization of mice with the T/B cell peptides produced high titered antibodies against the VD I sequence, and these antibodies reacted with the native MOMP and intact chlamydiae. The MOMP sequences containing these Th cell epitopes are conserved among the MOMP genes of different C. trachomatis serovars, indicating that they are common Th cell antigenic sites. Thus, the Th cell epitopes contained within these peptides, in combination with different trachoma serovar-specific B cell neutralizing determinants, may be useful in the development of a synthetic or recombinant trivalent trachoma vaccine.

Amino Acid Sequence↗

PubMed1991

Evidence that the major outer membrane protein of Chlamydia trachomatis is glycosylated.

The major outer membrane protein (MOMP) of Chlamydia trachomatis was determined to be a glycoprotein on the basis of susceptibility to glycosidase digestion and the presence of carbohydrate by staining and radiolabeling. The MOMP of the serovar L2 organisms was isolated by electroelution from the protein band excised from the gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The incubation of MOMP with N-glycosidase F, an endoglycosidase that cleaves the N-glycan, and periodate resulted in two new molecular weight species. While MOMP treated with N-glycosidase F showed a lower-molecular-weight mobility, the periodate-treated MOMP increased in molecular weight. Both treatments abolished the ability of the MOMP to bind to HeLa cell components. In the immunoblot, the reactivity to the monoclonal antibody specific against the C. trachomatis species was preserved. The endoglycosidase specific to O-linked glycan, endo-alpha-N-acetylgalactosaminidase, had no visible effect on the isolated MOMP. Carbohydrate was detected in the MOMP by p-phenylenediamine staining of the protein band in the gel following SDS-PAGE. Autoradiograms of proteins of chlamydial organisms metabolically labeled with [3H]galactose or [3H]glucosamine and separated by SDS-PAGE revealed the MOMP band. The isolated MOMP was shown to bind specifically to concanavalin A, wheat germ agglutinin, and Dolichos biflorus agglutinin in the lectin binding assay. No binding was observed with Ulex europaeus agglutinin I, soybean agglutinin, or Ricinus communis agglutinin.

Animals↗

PubMed1990

Purification and characterization of two major outer membrane proteins from Porphyromonas gingivalis.

Outer membrane proteins (OMPs) were extracted from whole cells of several Porphyromonas and Prevotella strains and their OMPs profiles were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE analysis revealed that OMP profiles of Porphyromonas and Prevotella strains show species-specific patterns and P. gingivalis characteristically had two kinds of major outer membrane proteins (MOMPs). A 53 Kd MOMP from P. gingivalis FDC 381 and a 67 Kd MOMP from ATCC 33277 were purified. Sera from periodontitis patients and healthy subjects were analyzed for immunoreactivities against both the purified MOMPs of P. gingivalis by immunoblotting analysis. The sera from 18 patients reacted to the 53 Kd MOMP, 10 to the 67 Kd MOMP, and only three sera reacted to both MOMPs. The sera of healthy subjects also reacted, but weakly, to either the 53 Kd or 67 Kd MOMP. The SDS-PAGE OMP profiles prepared from 13 clinical isolates of P. gingivalis and immunoblotting analysis of human sera against the two kinds of P. gingivalis MOMPs indicate that periodontal diseases resulting from P. gingivalis are initiated and sustained by at least two MOMPs of P. gingivalis.

Bacterial Outer Membrane Proteins↗

PubMed1989

Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane proteins of the 15 Chlamydia trachomatis serovars.

The amino acid sequences of major outer membrane proteins (MOMPs) from Chlamydia trachomatis serovars A, B, C, L1, and L2 are predominantly conserved but have four variable domains (VDs) in which major neutralizing and serotyping antigenic determinants are located. Because these MOMP VDs are primarily responsible for antigenic differences between serovars and are associated with important immunological and biological properties, we undertook studies focused on defining these sequences within the MOMPs of all 15 C. trachomatis serovars. We used oligonucleotide primer extension sequencing of MOMP mRNA to determine the nucleotide and deduced amino acid sequences of the four MOMP VDs of the 15 C. trachomatis serovars. Comparative amino acid sequence homologies of all four domains separated the serovars into three groups: group 1, serovars B, Ba, D, E, L1, and L2; group 2, serovars G and F; and group 3, serovars A, C, H, I, J, K, and L3. Hydrophilicity and charge values for each domain were determined. The MOMP VDs of given serovars with the greatest total hydrophilicity and charge values were found to be the location of antigenic determinants recognized by MOMP-specific monoclonal antibodies. These findings should be useful for predicting MOMP antigenic determinants and testing the antigenic properties of these VDs by using synthetic peptides corresponding to each MOMP VD. The potential usefulness of the VD sequence information is discussed in relation to the development of defined synthetic peptides and oligonucleotides that may be used to develop new serological and diagnostic assays for C. trachomatis infections.

Amino Acid Sequence↗

PubMed1988

Chemical cross-linking of Chlamydia trachomatis.

Purified elementary bodies (EBs) of Chlamydia trachomatis serovar L2 were analyzed by chemical cross-linking with disuccinimidyl selenodipropionate. The effect of the cross-linking was analyzed by immunoblotting sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated components which were reacted with monoclonal antibodies against major outer membrane protein (MOMP) and lipopolysaccharide (LPS). It was shown that in EBs, MOMP was cross-linked to the LPS component of the outer membrane. Migration analysis of the cross-linked components showed that with extensive cross-linking, most of the MOMP became cross-linked to LPS, changing the migration rate from 40 to 42.5 kilodaltons. A small fraction of MOMP associated with LPS was shown to be present in bands with migration rates of 100 and 110 kilodaltons. No association of MOMP or LPS to other proteins, or to dimer or multimer forms of MOMP without LPS, was observed. A totally different membrane structure must be present in reticulate bodies, since there, MOMP was so heavily cross-linked that it did not enter the polyacrylamide gel and thus became impossible to analyze. Furthermore, the monoclonal antibody, which reacted with LPS associated with MOMP in the cross-linked EBs, did not react with reticulate bodies.

Antibodies, Monoclonal↗

PubMed1989

Cloning and sequence analysis of the major outer membrane protein genes of two Chlamydia psittaci strains.

We cloned and sequenced the gene encoding the major outer membrane protein (MOMP) of two Chlamydia psittaci strains, guinea pig inclusion conjunctivitis (GPIC) strain 1, and meningopneumonitis (Mn) strain Cal-10. Intraspecies alignment of the two C. psittaci MOMP genes revealed 80.6% similarity, and interspecies comparison of C. trachomatis and C. psittaci MOMP genes yielded about 68% similarity. As found previously for C. trachomatis MOMP sequences, stretches of predominantly conserved sequences of GPIC and Mn MOMPs were interrupted by four variable domains whose locations were identical to those of C. trachomatis MOMPs. Seven of eight cysteine residues were found at precisely the same positions in GPIC, Mn, and C. trachomatis MOMPs, emphasizing their importance in structure and function of the protein. Collectively, these results indicate that C. psittaci and C. trachomatis MOMP genes diverged from a common ancestor.

Amino Acid Sequence↗

PubMed1990

Chlamydia trachomatis-host cell interactions: role of the chlamydial major outer membrane protein as an adhesin.

The major outer membrane protein (MOMP) of Chlamydia trachomatis is characterized by four symmetrically spaced variable domains (VDs I to IV) whose sequences vary among serotypes. The surface-exposed portions of these VDs contain contiguous sequences that are both serotyping determinants and in vivo target sites for neutralizing antibodies. Previous studies using surface proteolysis of C. trachomatis B implicated VDs II and IV of the MOMP of this serotype in the attachment of chlamydiae to host cells. In this study, we used monoclonal antibodies (MAbs) specific to antigenic determinants located in VDs II and IV of the MOMP of serotype B to further investigate the role of the MOMP in the attachment of chlamydiae to host cells. MABs specific to serotype- and subspecies-specific epitopes located in exposed VDs II and IV, respectively, neutralized chlamydial infectivity for hamster kidney cells by blocking chlamydial attachment. We radioiodinated these MAbs and used them to determine the number and topology of the surface-exposed VDs II and IV epitopes on chlamydial elementary bodies. VDs II and IV each comprised approximately 2.86 x 10(4) negatively charged sites and were in proximity on the chlamydial cell surface. These studies suggest that the MAbs blocked chlamydial attachment by inhibiting electrostatic interactions with host cells. We examined the effects of thermal inactivation on both chlamydial attachment and conformation of the MOMP. Heat-inactivated chlamydiae failed to attach to host cells and exhibited a conformational change in an inaccessible invariant hydrophobic nonapeptide sequence located within VD IV of the MOMPs of C. trachomatis serotypes. These findings suggest that in addition to electrostatic interactions, a common hydrophobic component of the MOMP also contributes to the binding of chlamydiae to host cells. Thus, we propose that the MOMP functions as a chlamydial adhesin by promoting nonspecific (electrostatic and hydrophobic) interactions with host cells. Surface-accessible negatively charged VDs appear to be important in electrostatic binding, while the invariant region of VD IV may provide a subsurface hydrophobic depression which further promotes binding of chlamydiae to host cells through hydrophobic interactions.

Antibodies, Monoclonal↗

PubMed1990

Cyclic AMP inhibits protein synthesis in Chlamydia trachomatis at a transcriptional level.

Cyclic AMP (cAMP) has an inhibitory effect on the developmental cycle of Chlamydia trachomatis. We examined its influence on the synthesis of chlamydial protein, using the major outer membrane protein (MOMP) as a marker for general chlamydial protein synthesis. During normal development MOMP synthesis accelerates from 18 h post-infection and peaks by 36 h. Cyclic AMP blocks this normal progression of the chlamydial growth cycle. At a concentration of 1 mM, nearly 75% of the total MOMP synthesis was inhibited by 36 h, as monitored by radiolabel uptake. However, no difference was observed during the first 12 h between cAMP-treated and control groups, a finding which is in keeping with correlation between developmental inhibition and protein synthesis. Hybridization studies carried out with a cloned MOMP gene demonstrate a drastic decrease in MOMP mRNA in cAMP-treated cells. Low levels of cAMP utilized in conjunction with a 100,000 x g supernatant from reticulate bodies (RBs) blocked the transcription of the recombinant MOMP gene in an in vitro transcription system. These results suggest that the inhibition of chlamydial protein synthesis, assessed by MOMP synthesis, is due to regulation at a transcriptional level.

Bacterial Outer Membrane Proteins↗

PubMed1985

Effect of proteolytic cleavage of surface-exposed proteins on infectivity of Chlamydia trachomatis.

The proteolytic cleavage of Chlamydia trachomatis LGV-434 surface proteins and resultant effects on infectivity and association with cultured human epithelial (HeLa) cells have been examined. Of several proteases examined, trypsin, chymotrypsin, and thermolysin extensively cleaved the chlamydial major outer membrane protein (MOMP). Two proteases, trypsin and thermolysin, cleaved the MOMP to the extent that monomeric MOMP was not detectable by immunoblotting with monospecific polyclonal antibodies. In the case of thermolysin, not even antigenic fragments were detected. Surprisingly, infectivity toward HeLa cells was not diminished. In addition, the association of intrinsically 14C-radiolabeled elementary bodies (EBs) with HeLa cells or their dissociation by proteinase K was not measurably affected by prior trypsinization of the EBs. Trypsinization of lactoperoxidase surface-iodinated elementary bodies demonstrated that most of the 125I-labeled surface proteins were cleaved. In all cases, however, a number of proteolytic cleavage fragments remained associated with the EB surface after surface proteolysis. When trypsinized EBs were electrophoresed under nonreducing conditions and immunoblotted with either polyclonal or type-specific monoclonal MOMP antibodies, MOMP was found in a large oligomeric form that failed to enter the polyacrylamide stacking gel. Additionally, trypsinized viable EBs bound radioiodinated type-specific MOMP monoclonal antibody as efficiently as did the control nontrypsinized organisms. Taken together, the findings indicate that although the MOMP is highly susceptible to surface proteolysis, the supramolecular structure of the protein on the EB surface is apparently maintained by disulfide interactions. Thus, if surface-exposed chlamydial proteins are involved in the initial interaction of chlamydiae with eucaryotic cells, the functional domains of these proteins which mediate this interaction must be resistant to proteolysis and remain associated with the EB surface.

Antigens, Bacterial↗

PubMed1986

Antigenic analysis of the major outer membrane protein of Chlamydia trachomatis with murine monoclonal antibodies.

We prepared monoclonal antibodies against prototype strains of the 15 serovars of Chlamydia trachomatis and identified a subset of reagents that reacted with the major outer membrane protein(s) (MOMPs) of one or more serovars. We then determined the specificities of these anti-MOMP monoclonal antibodies by radioimmunoassay and immunoblot assays against the 15 serovars of C. trachomatis and a C. psittaci strain. We identified 14 different anti-MOMP antibody specificities, including serovar-, several orders of subspecies-, and species-specific determinants. In addition, one antibody reacted with all C. trachomatis serovars and a C. psittaci strain, indicating the presence of a genus-specific epitope on MOMP. Many of the cross-reactions of the subspecies-specific antibodies were similar to those previously reported by use of the microimmunofluorescence technique. We also observed a number of cross-reactions that were unexpected but consistent with data derived by the microimmunofluorescence test. All antibodies, except the genus-specific antibodies, reacted with whole elementary bodies in a radioimmunoassay, suggesting surface exposure of the epitopes. These data confirm and extend previous observations that MOMPs among C. trachomatis serovars are antigenically complex and diverse. In addition, these data indicate that the cross-reaction patterns of some monoclonal antibodies directed against MOMP are similar to those detected by the microimmunofluorescence test and are consistent with the hypothesis that such determinants are contained within MOMPs.

Animals↗

PubMed1991

Sequence analysis of the major outer membrane protein gene of Chlamydia pneumoniae.

Compared with the major outer membrane proteins (MOMPs) of the other chlamydial species, the Chlamydia pneumoniae MOMP appears to be less antigenically complex, and as determined by immunoblot analysis, it does not appear to be the immunodominant antigen recognized during infection. Nucleotide sequence analysis of the C. pneumoniae MOMP gene (ompA) revealed that it consisted of a 1,167-base open reading frame with an inferred 39,344-dalton mature protein of 366 amino acids plus a 23-amino-acid leader sequence. A ribosomal-binding site was located in the 5' upstream region, and two stop codons followed by an 11-base dyad forming a stable stem-loop structure were identified. This sequence shares 68 and 71% DNA sequence homology to the Chlamydia trachomatis serovar L2 and Chlamydia psittaci ovine abortion agent MOMP genes, respectively. Interspecies alignment identified regions, corresponding to the variable domains, which share little sequence similarity with the other chlamydial MOMPs. All seven cysteines conserved in the C. trachomatis and C. psittaci MOMPs, which are involved in the formation of disulfide cross-linkages, are found in the C. pneumoniae MOMP.

Amino Acid Sequence↗

PubMed1992

Major outer membrane proteins of Vibrio cholerae and their role in induction of protective immunity through inhibition of intestinal colonization.

Vibrio cholerae O1 organisms belonging to different biotypes and serotypes were shown to express major outer membrane proteins (MOMPs) with subunit molecular masses of 48 to 50, 40 to 43, 35 to 36, 27 to 28, and 20 kDa. Antisera raised against individual MOMPs of a V. cholerae O1 strain recognized MOMPs of corresponding molecular masses in other O1 and non-O1 strains. Serological data also suggested possible differences in the cell surface exposition of these MOMPs. However, no marked differences between V. cholerae cells grown in vitro and in vivo could be noted in respect to the expression or surface exposition of these MOMPs. Of five MOMPs studied in this work, 40- to 43- and 20-kDa cell surface proteins were shown to be of considerable importance, as antisera to these proteins induced significant protection against V. cholerae challenge in the suckling mouse model. Similar protection, although to a lesser extent, was demonstrable with the antiserum to the 27- to 28-kDa protein. These results were corroborated with the Fab (immunoglobulin G) [Fab(IgG)] fragments of the antisera, thereby suggesting that the observed protection induced by anti-MOMP antibodies did not arise as a result of bacterial clumping. Subsequent studies demonstrated that these antisera as well as their Fab (IgG) fragments induced significant inhibition of intestinal colonization of V. cholerae. The 40- to 43- and 27- to 28-kDa proteins appeared to be porinlike, while the 20-kDa protein was found to be antigenically related to TcpA (subunit A of toxin-coregulated pilus). All these results demonstrate the involvement of more than one cell surface antigen of V. cholerae in the induction of protective immunity through inhibition of intestinal colonization of vibrios.

Animals↗

PubMed1992

Immune specificity of murine T-cell lines to the major outer membrane protein of Chlamydia trachomatis.

The antigenically variant Chlamydia trachomatis major outer membrane protein (MOMP) is a target of antibody-mediated neutralization in vitro, and it is an important protein for designing a subunit vaccine. Knowledge of MOMP T-cell determinants will be essential to elicit rapid and strong immune responses following an encounter with infectious organisms. C. trachomatis-specific T-cell lines were derived from MOMP-immunized BALB/c mice and selected with intact organisms. We used these short-term T-cell lines to identify determinants of MOMP that could be recognized by T cells following processing of the intact organism. T-cell line proliferation in response to overlapping MOMP peptides showed that only a peptide encompassing the third variable segment (VS3) elicited a strong proliferative response. We further mapped determinants within the VS3 peptide and found that a sequence-conserved portion of the VS3 peptide elicited T-cell proliferation of T-cell lines from BALB/c mice. Thus, unlike the response to several MOMP peptides with unselected T cells, development of short-term T-cell lines with intact organisms restricted the repertoire of antigens capable of being recognized by MOMP-specific T cells.

Amino Acid Sequence↗

PubMed1992

Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp.

Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis.

Animals↗