Immunobiology of purified recombinant outer membrane porin protein I of Neisseria gonorrhoeae

Gonococcal porins (Por) from strains FA19 (Por-1, serogroup A), MS11 (Por-2, serogroup B) and FA6434 (Por-5, a hybrid porin containing epitopes from both serogroups), were expressed in Escherichia coli and purified under non-denaturing conditions. Porins were inserted into liposomes, and they were bound by monoclonal antibodies which bind native Por and intact gonococci, but not denatured Por. All three recombinant porins (rPor) were highly immunogenic in rabbits without additional adjuvant. The rPor antisera were specific for Por by Western blotting and whole-cell radioimmunoprecipitation and were broadly cross-reactive within serogroups. Post-immune, but not pre-immune, sera bound to intact gonococci, induced deposition of complement components C3 and C9 onto gonococcal membranes and increased association with and activation of human neutrophils. Gonococci were not killed in bactericidal assays, and there was no phagocytic killing with gonococci opsonized with recombinant antisera. Lack of killing in bactericidal assays was not caused by the presence of blocking antibodies to the outermembrane protein Rmp.     

Monoclonal antibodies to gonococcal outer membrane protein I: location of a conserved epitope on protein IB

Hybrid cell lines have been derived which produce monoclonal antibodies reacting with outer membrane protein I from Neisseria gonorrhoeae strain P9. The antibodies obtained showed variable reactivity with other strains but one antibody recognized an epitope present on all of the strains tested which expressed the protease sensitive protein IB. Purified IgG labelled with 125I was used in competitive radioimmunoassays with unlabelled antibody to investigate the spacial distribution of the epitopes recognized. Each pair of antibodies showed some degree of inhibition. The relative magnitude of inhibition suggested that the conserved epitope lies within a variable region containing other epitopes which determine the antigenic specificity of the protein. Western blotting of peptides derived by proteolytic digestion of protein IB revealed that the conserved epitope is located close to the chymotrypsin cleavage site within a 7000 Mr surface exposed region of the molecule.     

Antigenic and immunogenic properties of cyanogen bromide peptides from a serotype 5 gonococcal outer membrane protein I

Treatment of a gonococcal major outer membrane protein IB (serotype 5) with cyanogen bromide (CNBr) resulted in cleavage of PIB into three major fragments of apparent molecular weight of 15, 13, and 8 kD. The location of these peptides in the intact protein was determined by analysis of partial cleavage products. The 8 kD peptide (CB2) was found to be located in the central region of the protein. Chymotrypsin cleavage of PIB revealed a cleavage site near one of the CNBr cleavage sites. Trypsin was found to cleave the protein, either in outer membranes (OMC) or in detergent micelles, in the central CB2 fragment. These result suggest that CB2 is a part of the surface exposed region of PIB.Immunization of mice with purified PIB (serotype 5) induced antibodies against all three CB-peptides. Absorption of the sera with homologous intact OMC resulted in a complete removal of antibodies against CB2, supplying further evidence for its surface exposed nature. Antibodies against the 13 kD peptide (CB1) could not be absorbed with intact OMC, suggesting that this peptide is buried within the outer membrane.Antisera raised against CB2 of serotype 5 demonstrated a considerable cross-reactivity with heterologous outer membranes. On the contrary, intact OMC induced mainly type-specific antibodies. These data demonstrate the presence of conserved epitopes on the surface exposed CB2 peptide. These conserved epitopes are generally not very immunogenic when present in intact OMC.     

Gene polymorphism analysis of Yersinia enterocolitica outer membrane protein A and putative outer membrane protein A family protein

Background

Yersinia enterocolitica outer membrane protein A (OmpA) is one of the major outer membrane proteins with high immunogenicity. We performed the polymorphism analysis for the outer membrane protein A and putative outer membrane protein A (p-ompA) family protein gene of 318 Y. enterocolitica strains.

Results

The data showed all the pathogenic strains and biotype 1A strains harboring ystB gene carried both ompA and p-ompA genes; parts of the biotype 1A strains not harboring ystB gene carried either ompA or p-ompA gene. In non-pathogenic strains (biotype 1A), distribution of the two genes and ystB were highly correlated, showing genetic polymorphism. The pathogenic and non-pathogenic, highly and weakly pathogenic strains were divided into different groups based on sequence analysis of two genes. Although the variations of the sequences, the translated proteins and predicted secondary or tertiary structures of OmpA and P-OmpA were similar.

Conclusions

OmpA and p-ompA gene were highly conserved for pathogenic Y. enterocolitica. The distributions of two genes were correlated with ystB for biotype 1A strains. The polymorphism analysis results of the two genes probably due to different bio-serotypes of the strains, and reflected the dissemination of different bio-serotype clones of Y. enterocolitica.     

Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles

Acinetobacter baumannii is an important nosocomial pathogen that causes a high morbidity and mortality rate in infected patients, but pathogenic mechanisms of this microorganism regarding the secretion and delivery of virulence factors to host cells have not been characterized. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs) that play a role in the delivery of virulence factors to host cells. A. baumannii has been shown to secrete OMVs when cultured in vitro, but the role of OMVs in A. baumannii pathogenesis is not well elucidated. In the present study, we evaluated the secretion and delivery of virulence factors of A. baumannii to host cells via the OMVs and assessed the cytotoxic activity of outer membrane protein A (AbOmpA) packaged in the OMVs. A. baumannii ATCC 19606(T) secreted OMVs during in vivo infection as well as in vitro cultures. Potential virulence factors, including AbOmpA and tissue-degrading enzymes, were associated with A. baumannii OMVs. A. baumannii OMVs interacted with lipid rafts in the plasma membranes and then delivered virulence factors to host cells. The OMVs from A. baumannii ATCC 19606(T) induced apoptosis of host cells, whereas this effect was not detected in the OMVs from the ΔompA mutant, thereby reflecting AbOmpA-dependent host cell death. The N-terminal region of AbOmpA(22-170) was responsible for host cell death. In conclusion, the OMV-mediated delivery of virulence factors to host cells may well contribute to pathogenesis during A. baumannii infection.     

Acinetobacter baumannii outer membrane protein A modulates the biogenesis of outer membrane vesicles

Acinetobacter baumannii secretes outer membrane vesicles (OMVs) during both in vitro and in vivo growth, but the biogenesis mechanism by which A. baumannii produces OMVs remains undefined. Outer membrane protein A of A. baumannii (AbOmpA) is a major protein in the outer membrane and the C-terminus of AbOmpA interacts with diaminopimelate of peptidoglycan. This study investigated the role of AbOmpA in the biogenesis of A. baumannii OMVs. Quantitative and qualitative approaches were used to analyze OMV biogenesis in A. baumannii ATCC 19606T and an isogenic ΔAbOmpA mutant. OMV production was significantly increased in the ΔAbOmpA mutant compared to wild-type bacteria as demonstrated by quantitation of proteins and lipopolysaccharides (LPS) packaged in OMVs. LPS profiles prepared from OMVs from wild-type bacteria and the ΔAbOmpA mutant had identical patterns, but proteomic analysis showed different protein constituents in OMVs from wild-type bacteria compared to the ΔAbOmpA mutant. In conclusion, AbOmpA influences OMV biogenesis by controlling OMV production and protein composition.     

Arrangement of protein I in Escherichia coli outer membrane: cross-linking study.

The arrangement of protein I in the outer membrane of Escherichia coli was investigated by cross-linking whole cells, isolated cell wall, protein-peptidoglycan complexes, and protein I released from peptidoglycan with NaCl. Both cleavable azide cross-linkers and imidoester reagents were used. The data presented suggest that protein I exists in the outer membrane as a trimer.     

A common-immunogenic Vibrio outer membrane protein

Abstract The presence of antibodies in rabbit antisera to cell envelope proteins of Vibrio cholerae has been examined using a two-dimensional system, in which the cell envelopes are electrophoresed in sodium dodecyl sulfate (SDS) in polyacrylamide gels in the first dimension, and in agarose containing antibodies in the second. The results show that a 25-kDa protein is markedly immunogenic and appears to be common to Vibrio strains; it is not present in a number of other organisms. This 25-kDa protein is an outer membrane protein as judged by sucrose gradient centrifugation and it is accessible to iodination by lactoperoxidase, suggesting that it is exposed on the cell surface.     

Insights into outer membrane protein crystallization

Outer membrane proteins are structurally distinct from those that reside in the inner membrane and play important roles in bacterial pathogenicity and human metabolism. X-ray crystallography studies on >40 different outer membrane proteins have revealed that the transmembrane portion of these proteins can be constructed from either β-sheets or less commonly from α-helices. The most common architecture is the β-barrel, which can be formed from either a single anti-parallel sheet, fused at both ends to form a barrel or from multiple peptide chains. Outer membrane proteins exhibit considerable rigidity and stability, making their study through x-ray crystallography particularly tractable. As the number of structures of outer membrane proteins increases a more rational approach to their crystallization can be made. Herein we analyse the crystallization data from 53 outer membrane proteins and compare the results to those obtained for inner membrane proteins. A targeted sparse matrix screen for outer membrane protein crystallization is presented based on the present analysis.     

The class 1 outer membrane protein of Neisseria meningitidis: gene sequence and structural and immunological similarities to gonococcal porins

The class 1 protein is a major protein of the outer membrane of Neisseria meningitidis, and an important immunodeterminant in humans. The complete nucleotide sequence for the structural gene of a class 1 protein has been determined. The sequence predicts a protein of 374 amino acids, preceded by a typical signal peptide of 19 residues. The hydropathy profile of the predicted protein sequence resembles that of the Escherichia coli and gonococcal porins. The predicted protein sequence of the class 1 protein exhibits considerable structural similarity to the gonococcal porins PIA and PIB. Western blot studies also reveal immunologically conserved domains between the class 1 protein, PIA and PIB. A restriction fragment from the class 1 gene hybridizes to gonococcal genomic fragments in Southern blots. In addition to the class 1 gene coding region there is a large open reading frame on the opposite strand.     

Chloroplast outer membrane protein targeting and insertion

Proteins in the chloroplast outer envelope membrane are nuclear encoded and post-translationally targeted to the chloroplast. The targeting and membrane insertion of these proteins is not well understood. Although early work suggested otherwise, the best-studied outer membrane proteins (OMPs) use both proteins within the chloroplast and NTPs for insertion. There have been conflicting reports in the field regarding protein targeting and insertion, which have probably arisen because of differences in experimental methodology and different interpretations of reduction (versus abolition) of integration. This review summarizes what is known to date about the mechanism of chloroplast OMP targeting.     

The class 3 outer membrane protein (PorB) of Neisseria meningitidis: gene sequence and homology to the gonococcal porin PIA

The class 3 protein (PorB) is an important component of the meningococcal outer membrane. The structural gene (porB) encoding the class 3 protein has been cloned using primers suitable for the amplification of the corresponding chromosomal fragment by the polymerase chain reaction (PCR). The complete nucleotide sequence was determined and predicts a mature protein of 310 amino acids, preceded by a signal peptide of 19 residues. The predicted protein sequence of the class 3 protein exhibits essential structural homology to the gonococcal porin PIA. The class 3 protein encoding gene was expressed in Escherichia coli under the control of an inducible promoter.     

Deoxyribonucleic acid and outer membrane: binding to outer membrane involves a specific protein.

The binding of deoxyribonucleic acid (DNA) to the outer membrane of Escherichia coli was examined. The amount of DNA found to be bound to outer membrane was low and was estimated to be about 0.4% of the total DNA. Treatment of cells with chloramphenicol or rifampin caused a disassociation of the apparent DNA-outer membrane complex. The results presented here suggest that the binding between membrane and DNA is specific and involves a membrane protein having a molecular weight of 13,000.     

Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia.

The outer membrane of Spirochaeta aurantia was isolated after cells were extracted with sodium lauryl sarcosinate and was subsequently purified by differential centrifugation and KBr isopycnic gradient centrifugation. The purified outer membrane was obtained in the form of carotenoid-containing vesicles. Four protein species with apparent molecular weights of 26,000 (26K), 36.5K, 41K, and 48.5K were readily observed as components of the vesicles. The 36.5K protein was the major polypeptide and constituted approximately 90% of the outer membrane protein observed on sodium dodecyl sulfate-polyacrylamide gels. Under mild denaturing conditions the 36.5K major protein exhibited an apparent molecular weight of approximately 90,000. This, together with the results of protein cross-linking studies, indicates that the 36.5K polypeptide has an oligomeric conformation in the native state. Reconstitution of solubilized S. aurantia outer membrane into lipid bilayer membranes revealed the presence of a porin, presumably the 36.5K protein, with an estimated channel diameter of 2.3 nm based on the measured single channel conductance of 7.7 nS in 1 M KCl.     

Cross-linking analysis of the two forms of protein I, a major outer membrane protein of Escherichia coli.

The two forms of protein I were cross-linked to molecules of the same species, even when both were present simultaneously. This suggests that they form separate multimers in the outer membrane.     

Bipartite gating in the outer membrane protein FecA

The recent structure determination of FecA, with and without ligand, shows the existence of two gates. These are the extracellular loops closing over the binding site and the plug located inside the barrel. It indicates a process which is described as bipartite gating and allows for a rational distinction between the binding event and the transport process.     

Mechanisms of protein import across the mitochondrial outer membrane

Mitochondria import the majority of their proteins from the cytosol. At the mitochondrial outer membrane, import is initiated through a series of reactions, which include preprotein recognition, unfolding, insertion and translocation. These processes are facilitated by a multisubunit complex, the TOM complex. Specific roles can now be assigned to several components of this complex. Although the import machinery of the outer membrane can insert and translocate a few proteins on its own, completion of translocation o f most preproteins is dependent upon coupling to both the membrane potential and mt-Hsp70/ATP-driven transport across the inner membrane, mediated by the TIM complex.