Polymorphic Outer Membrane Protein Research Articles

Identification of Chlamydia pneumoniae-derived mouse CD8 epitopes.

Chlamydia pneumoniae is a common intracellular human pathogen that has been associated with several severe pathological conditions, including coronary heart disease and atherosclerosis. There is no vaccine against C. pneumoniae infection, but CD8(+) T cells have been shown to be crucial for protection during experimental infection. However, the effector functions and epitope specificity of the protective CD8(+) T cell remain unknown. The aim of this study was to identify C. pneumoniae-derived mouse CD8 epitopes by using a recent epitope prediction method. Of four C. pneumoniae proteins (the major outer membrane protein, outer membrane protein 2, polymorphic outer membrane protein 5, and heat shock protein 60), 53 potential CD8(+) T-cell epitopes were predicted by H-2 class I binding algorithms. Nineteen of the 53 peptides were identified as CD8 epitopes by testing for induction of a cytotoxic response after immunization. To test whether the predicted epitopes are naturally processed and presented by C. pneumoniae-infected cells, we generated a panel of seven peptide-specific cytotoxic T lymphocyte lines that were subsequently tested for recognition of C. pneumoniae-infected target cells. By using this strategy, we were able to identify three C. pneumoniae CD8 epitopes that were, indeed, processed and presented on infected cells. Identification of these natural CD8 epitopes provides tools for characterization of CD8(+) T-cell function in vivo and generation of epitope-specific prevention strategies.

The expression, processing and localization of polymorphic membrane proteins in Chlamydia pneumoniae strain CWL029

BackgroundChlamydiae are obligate intracellular bacteria, which are important human pathogens. Genome sequences of C. trachomatis and C. pneumoniae have revealed the presence of a Chlamydia specific gene family encoding polymorphic outer membrane proteins, Pmps. In C. pneumoniae the family comprises twenty-one members, which are all transcribed. In the present study, the expression, processing and localisation of the sixteen full-length Pmps in C. pneumoniae strain CWL029 have been further investigated by two-dimensional gel electrophoresis and immunofluorescence microscopy.ResultsTen Pmps were identified in elementary bodies (EBs). Eight of these were investigated with respect to time dependent expression and all were found to be up-regulated between 36 and 48 hours post infection. Antibodies against Pmp6, 8, 10, 11 and 21 reacted with chlamydiae when infected cells were formalin fixed. Pmp6, Pmp20 and Pmp21 were found in cleaved forms, and the cleavage sites of Pmp6 and Pmp21 were identified.ConclusionsThe Pmps are heavily up-regulated at the time of conversion of RB to EB, and at least ten Pmps are present in EBs. Due to their reaction in formalin fixation it is likely that Pmp6, 8, 10, 11 and 21 are surface exposed. The identified cleavage sites of Pmp6 and Pmp21 are in agreement with the theory that the Pmps are autotransporters.

Polymorphic outer-membrane proteins of Chlamydophila abortus are glycosylated.

Antigenic profiles of mono-, bi- and poly-specific monoclonal antibodies against 90 kDa polymorphic outer-membrane proteins (POMPs) and a 105 kDa POMP-related protein of Chlamydophila abortus ATCC VR 656(T), after one- and two-dimensional electrophoretic analysis, helped identify each one of the triplets POMP 90, 91A and 91B, and a POMP-related protein at 85 kDa. The lectin concanavalin A bound to the four POMPs and the POMP-related protein in a specific manner and the binding was sensitive to treatment with the amidase N-endoglycosidase F, suggesting the presence of small asparagine-linked oligosaccharide chains. The exposure of the five proteins on the chlamydial surface and the orientation of the attached oligosaccharide chains was examined by protease and endoglycosidase treatments of intact bacteria. The results were consistent with the concept that some of the oligosaccharides in the POMPs face outwards, possibly protecting the polypeptides from proteolytic enzymes, whereas the oligosaccharides in the 105 kDa POMP-related protein are oriented inwards, thereby rendering the polypeptide chain accessible to proteases. A possible role for the N-linked oligosaccharides in the POMPs might be the promotion of the proper folding and processing of these proteins.

Characterization of outer membrane proteins in Chlamydia trachomatis LGV serovar L2.

We used a photoactivatable, lipophilic reagent, 3'-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, to label proteins in the outer membrane of elementary bodies of Chlamydia trachomatis LGV serovar L2 and mass spectrometry to identify the labeled proteins. The identified proteins were polymorphic outer membrane proteins E, G, and H, which were made late in the developmental cycle, the major outer membrane protein, and a mixture of 46-kDa proteins consisting of the open reading frame 623 protein and possibly a modified form of the major outer membrane protein.

Identification of polymorphic outer membrane proteins of Chlamydia psittaci 6BC.

The genomes of Chlamydia spp. encode a family of putative outer membrane proteins, referred to as polymorphic outer membrane proteins (POMPs), which may play a role in the avoidance of host immune defenses. We analyzed avian strain 6BC of Chlamydia psittaci by polyacrylamide gel electrophoresis for the expression of POMPs. At least six putative POMPs were identified on the basis of their size (90 to 110 kDa) and labeling with an outer membrane-specific probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Three of the putative POMPs reacted with antiserum raised against a recombinant ovine C. psittaci strain POMP, and two possessed surface-exposed, trypsin-sensitive sites. The POMPs were dependent on disulfide bonds for their maintenance in sodium lauryl sarcosine- and sodium dodecyl sulfate-insoluble complexes but did not appear to be interpeptide disulfide bond cross-linked. The putative POMPs were found to be synthesized during the late phase of the chlamydial developmental cycle, cotemporally with the cysteine-rich doublet periplasmic proteins.

Diagnosis of ovine enzootic abortion using an indirect ELISA (rOMP91B iELISA) based on a recombinant protein fragment of the polymorphic outer membrane protein POMP91B of Chlamydophila abortus

Chlamydophila abortus is of major economic importance worldwide as one of the principal causes of abortion in sheep. Serological diagnosis of infection by the complement fixation test (CFT) is complicated by false positive reactions resulting from cross-reactive antibodies to Chlamydophila pecorum. To improve diagnosis an indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant protein fragment of the C. abortus polymorphic outer membrane protein POMP91B (rOMP91B iELISA) was assessed using a panel of 281 sera from experimentally and naturally infected sheep. The iELISA performed well, being more sensitive (84.2%) and specific (98.5%) than the CFT. Furthermore, the iELISA was better at differentiating C. abortus- from C. pecorum-infected animals. The new rOMP91B iELISA test will prove a valuable tool for the routine serodiagnosis of C. abortus infection.

Antimicrobial susceptibility of Haemophilus influenzae in the respiratory tracts of patients with cystic fibrosis.

We analyzed the antimicrobial susceptibilities of Haemophilus influenzae isolates from 157 sputum specimens prospectively collected from 39 cystic fibrosis (CF) patients during a 2-year study. These isolates were characterized by random amplified polymorphic DNA analysis and major outer membrane protein (MOMP) analysis to identify H. influenzae strains and MOMP variants and to assess their persistence in the respiratory tract. Among the 247 H. influenzae isolates, 16 (6.5%) produced beta-lactamase. The 231 beta-lactamase-negative isolates represented 85 H. influenzae strains, 61 MOMP variants derived from 27 of these strains, and 85 persistent isolates identical to strains or MOMP variants. All beta-lactamase-negative isolates were tested for susceptibility to ampicillin, amoxicillin-clavulanic acid, cefuroxime, cefotaxime, cefaclor, imipenem, tetracycline, and trimethoprim-sulfamethoxazole by disk diffusion testing. Eleven (13%) H. influenzae strains, 18 (30%) MOMP variants, and 30 (35%) persistent isolates were resistant to one or more of the antibiotics tested. Antimicrobial susceptibility was decreased among MOMP variants and persistent isolates compared to nonpersistent H. influenzae strains, and changes in susceptibility occurred irrespective of MOMP variation. We conclude that the decreased antimicrobial susceptibility of H. influenzae during persistence contributes to the poor eradication of H. influenzae from the respiratory tracts of CF patients.

The variable antigens Vmp7 and Vmp21 of the relapsing fever bacterium Borrelia hermsii are structurally analogous to the VSG proteins of the African trypanosome

The relapsing fever agent Borrelia hermsii avoids the host's immune response by the strategy of multiphasic antigenic variation. A given Borrelia cell can express one of a number of alleles for polymorphic outer-membrane proteins, known as Vmp proteins. The genes for the variant-specific Vmp proteins of serotypes 7 and 21 of B. hermsii strain HS1 were sequenced. The genes, which were designated vmp7 and vmp21, were obtained from populations of borreliae before and after a switch in serotypes from 7 to 21. The analysis showed that vmp7 and vmp21 are 77% identical in terms of their coding sequence. The deduced translation products of vmp7 and vmp21 are polypeptides of 369 (37.2 kD) and 364 amino acids (37.1 kD), respectively. Vmp7 and Vmp21 have sequence features of prokaryotic lipoproteins and are processed as such during expression in E. coli. The secondary structure predictions of the Vmp proteins reveals analogous structures to the VSG proteins of the African trypanosome.