Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139

Abstract The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the pateint. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.     

Expression and immunogenicity analysis of two iron-regulated outer membrane proteins of Vibrio parahaemolyticus

Genes of two iron-regulated outer membrane proteins of Vibrio parahaemolyticus zj2003, a pathogenic strain isolated from large yellow croaker （Pseudosciaena crocea）, psuA and pvuA, were cloned and expressed as N-terminal His6-tagged proteins in Escherichia coli BL21（DE3）. The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of the proteins, groups of large yellow croaker were immunized with the purified recombinant psuA, pvuA or both, by intraperitoneal injection. Antibody response was assessed by enzyme-linked immunosorbent assay. Titers to the recombinant proteins increased from log2 3.25 to log29.80, 4-8 weeks following immunization. The relative percent survival of the groups vaccinated with psuA, pvuA, or a combination of the two, reached 50%, 62.5% and 75%, respectively. Western blot analysis was carried out with the serum from unvaccinated survival fish after infection. Both recombinant proteins were detected, indicating that these two proteins of V. parahaemolyticus zj2003 were immunogenic and could produce synergistic effects during in vivo infection, and they might be considered as important components for developing an aquaculture vaccine against this pathogen.     

Comparative proteome analysis reveals pathogen specific outer membrane proteins of Leptospira

Proteomes of pathogenic Leptospira interrogans and L. borgpetersenii and the saprophytic L. biflexa were filtered through computational tools to identify Outer Membrane Proteins (OMPs) that satisfy the required biophysical parameters for their presence on the outer membrane. A total of 133, 130, and 144 OMPs were identified in L. interrogans, L. borgpetersenii, and L. biflexa, respectively, which forms approximately 4% of proteomes. A holistic analysis of transporting and pathogenic characteristics of OMPs together with Clusters of Orthologous Groups (COGs) among the OMPs and their distribution across 3 species was made and put forward a set of 21 candidate OMPs specific to pathogenic leptospires. It is also found that proteins homologous to the candidate OMPs were also present in other pathogenic species of leptospires. Six OMPs from L. interrogans and 2 from L. borgpetersenii observed to have similar COGs while those were not found in any intermediate or saprophytic forms. These OMPs appears to have role in infection and pathogenesis and useful for anti‐leptospiral strategies.     

Identification of heme-binding proteins in the cell membranes of Vibrio anguillarum

Abstract Two strains of Vibrio anguillarum belonging to O1 and O2 serotypes were examined for their ability to bind hemin and hemoglobin. Whole cells as well as membrane extracts from both strains could clearly bind hemin and hemoglobin constitutively. Hemoglobin binding was completely inhibited by a 100-fold excess of unlabelled hemoglobin and also by hemin, suggesting the existence of specific receptors for heme groups in the cell membranes. Several hemin-binding and hemoglobin-binding bands with similar molecular sizes were detected in polyacrylamide gels as well as in Western blots. Only two of these protein bands in both strains were iron-regulated while the others were independent of the cell iron status. We conclude that both serotypes of V. anguillarum possess heme-binding abilities by means of membrane proteins.     

Comparative proteome analysis of the outer membrane proteins of in vitro-induced multi-drug resistant Neisseria gonorrhoeae

Antimicrobial-resistant gonococcus has been a major problem in sexually transmitted disease control . Outer membrane proteins (OMPs) of Neisseria gonorrhoeae were suggested to have influence on its resistance to antibiotics. So, in this work, we provide a proteomic analysis tool for examining the OMPs of N. gonorrhoeae and also provide a comparative analysis of the OMPs between the susceptible parent strain (92WT) and the resistance-induced isogenic mutant (92mu13) to determine the OMPs responsible for resistance. The 2-D gel spots of 92mu13 differed from 92WT particularly in porin, pilus secretion protein (PilQ) and enzymes. PilQ expression in 92mu13 was considerably reduced by abrupt termination at nucleotide 2,112. This made it difficult to form a high molecular mass (HMM) pore at the outer membrane; it is suspected that reduction of PilQ serves a role in antibiotic resistance in N. gonorrhoeae. The amount of porin was not changed but its isoelectric point (pI) shifted to a basic region, which is caused by the alteration of an amino acid of porin and it is suggested to relate to the development of antimicrobial resistance . Differential regulation of the enzymes involved in metabolism was found in 92mu13, believed to represent an adaptation of N. gonorrhoeae to the antibiotic environment.     

Mutational analysis of the zinc metalloprotease EmpA of Vibrio anguillarum

The extracellular zinc metalloprotease, EmpA, is a putative virulence factor involved in pathogenicity of the fish pathogen Vibrio anguillarum. The 611-amino acid precursor of this enzyme is encoded by the empA gene. The residues His346, His350, Glu370, Glu347, His429, Tyr361 and Asp417 are highly conserved and putatively function together at the active site of the enzyme. In this study, empA was inserted into pET24d(+) and expressed in Escherichia coli strain BL21(DE3) as a 6 x His tagged protein (r-EmpA). All the conserved residues of EmpA mentioned above were individually mutated by site-directed mutagenesis and the mutants were also expressed (m-r-EmpAs). r-EmpA and m-r-EmpAs were purified, and assayed for their proteolytic activities with azocasein as the substrate and cytotoxicities on a flounder gill cell line. m-r-EmpAs that had been mutated at His346, His350, Glu370 and Glu347 almost completely lost their proteolytic activity and cytotoxicity, pointing towards the essential roles played by these residues. In contrast, those mutated at Tyr361, His429 and Asp417 still retained a partial proteolytic activity and cytotoxicity. Our results indicate that these conserved residues play important roles in enzymatic activity and that the proteolytic activity of the enzyme is involved in the pathogenesis of V. anguillarum     

Identification and evaluation of an outer membrane protein OmpU from a pathogenic Vibrio harveyi isolate as vaccine candidate in turbot (Scophthalmus maximus)

Aims: The main aims of this study were to clone and express a new outer membrane protein U (OmpU) from a pathogenic Vibrio harveyi SF‐1 and investigate its immune efficiency as a vaccine candidate against V. harveyi infection in turbot (Scophthalmus maximus). Methods and Results: In this study, a new gene, ompU was cloned from the genomic DNA of pathogenic V. harveyi SF‐1. The ompU gene encoded a 35 kDa protein, which was purified by Ni‐NTA His‐Bind Resin column. A DNA vaccine was constructed by inserting ompU gene into pEGFP‐N1 plasmid. Turbot were injected intramuscularly with the purified OmpU protein and the recombinant pEGFP‐N1/ompU plasmid, respectively. The fish vaccinated with the purified OmpU protein were completely protected with a relative per cent of survival (RPS) of 100% against pathogenic V. harveyi infection. Efficient protection was also found in the pEGFP‐N1/ompU vaccinated group, with a RPS of 51·4%. Significant specific antibody responses were detected in the vaccinated turbot by indirect enzyme‐linked immunosorbent assay. Conclusions: A new OmpU was cloned and expressed. Both OmpU protein vaccine and DNA vaccine showed good immune protections in turbot. Significance and Impact of the Study: The OmpU was identified to be a new effective vaccine candidate and could be used as subunit vaccine and DNA vaccine for disease control caused by pathogenic V. harveyi.     

Comparative immunogenicity of outer membrane protein K and whole-cell antigens of Vibrio parahaemolyticus for diagnosis

The immunogenicity of soluble outer membrane protein K (OmpK)- small ubiquitin-like modifier, OmpK inclusion bodies, formalin, and heat-killed Vibrio parahaemolyticus cells were prepared and studied in a mouse model. The results of whole-cell ELISA and Western blot (WB) revealed that the serum against soluble OmpK and OmpK inclusion bodies reacted only with homologous V. parahaemolyticus. Furthermore, recombinant OmpK proteins were not recognized by the serum against whole-cell V. parahaemolyticus antigens. Unexpectedly, the serum against formalin and heat-killed V. parahaemolyticus reacted broadly with homologous (an immunization strain) and heterologous (non-immunization strains) V. parahaemolyticus and Vibrio species. The WB results revealed that the serum against the two V. parahaemolyticus whole-cell antigens primarily reacted with proteins that were approximately 100, 70, 36, 28, and 22 kDa in the cell lysates from different Vibrio strains, rather than the recombinant OmpK. The 70 and 28 kDa proteins exhibited specificity to Vibrio species, while the 22 kDa protein was more specific to V. parahaemolyticus. This study showed the limitation of recombinant OmpK to prepare diagnostic antibodies and revealed several specific Omps of Vibrio sp. and V. parahaemolyticus that were promising in diagnosis and vaccine development.     

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo‐induced antigen technology (IVIAT). Methods and Results: The convalescent‐phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo‐induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion‐mutated, and the growth and 50% lethal dose (LD₅₀) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo‐expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.     

Multiple conformational states and gate opening of outer membrane protein TolC revealed by molecular dynamics simulations

Outer membrane protein TolC serves as an exit duct for exporting substances out of cell. The occluded periplasmic entrance of TolC is required to open for substrate transport, although the opening mechanism remains elusive. In this study, systematic molecular dynamics (MD) simulations for wild type TolC and six mutants were performed to explore the conformational dynamics of TolC. The periplasmic gate was shown to sample multiple conformational states with various degrees of gating opening. The gate opening was facilitated by all mutations except Y362F, which adopts an even more closed state than wild type TolC. The interprotomer salt‐bridge R367–D153 is turned out to be crucial for periplasmic gate opening. The mutations that disrupt the interactions at the periplasmic tip may affect the stability of the trimeric assembly of TolC. Structural asymmetry of the periplasmic gate was observed to be opening size dependent. Asymmetric conformations are found in moderately opening states, while the most and the least opening states are often more symmetric. Finally, it is shown that lowering pH can remarkably stabilize the closed state of the periplasmic gate. Proteins 2014; 82:2169–2179. © 2014 Wiley Periodicals, Inc.     

Comparison of outer membrane protein profiles of Vibrio vulnificus biotypes 1 and 2

Abstract The outer membrane proteins of 17 Vibrio vulnificus biotype 2 strains from Japanese and European cels, and 12 biotype 1 strains from clinical and environmental sources have been compared. The overall profile in both biotypes was similar, and a major protein band of molecular mass 36 kDa was detected in the majority of the strain. Differences in the minor bands allowed differentiation of strains from different origins, suggesting that outer membrane protein profiles could be useful as epidemiological markers in the species V. vulnificus . Immunoblotting with antisera to whole cells of selected strains of biotypes 1 and 2 showed a strong antigenic response to outer membrane proteins 66, 60, 48, 46 and 44 kDa; these were common to all strains examined, independent of their biotypes and origins. These results demonstrate the presence of antigenically related outer membrane proteins in both biotypes of V. vulnificus .     

Molecular analysis of VCA1008: a putative phosphoporin of Vibrio cholerae

The PhoB/PhoR-dependent response to inorganic phosphate (Pi)-starvation in Vibrio cholerae O1 includes the expression of vc0719 for the response regulator PhoB, vca0033 for an alkaline phosphatase and vca1008 for an outer membrane protein (OMP). Sequences with high identity to these genes have been found in the genome of clinical and environmental strains, suggesting that the Pi-starvation response in V. cholerae is well conserved. VCA1008, an uncharacterized OMP involved in V. cholerae pathogenicity, presents sequence similarity to porins of Gram-negative bacteria such as phosphoporin PhoE from Escherichia coli . A three-dimensional model shows that VCA1008 is a 16-stranded pore-forming β-barrel protein that shares three of the four conserved lysine residues responsible for PhoE anionic specificity with PhoE. VCA1008 β-barrel apparently forms trimers that collapse into monomers by heating. Properties such as heat modifiability and resistance to denaturation by sodium dodecyl sulfate at lower temperatures permitted us to suggest that VCA1008 is a classical porin, more precisely, a phosphoporin due to its Pi starvation-induced PhoB-dependent expression, demonstrated by electrophoretic mobility shift assay and promoter fusion- lacZ assays.     

New Escherichia coli outer membrane proteins identified through prediction and experimental verification

Many new Escherichia coli outer membrane proteins have recently been identified by proteomics techniques. However, poorly expressed proteins and proteins expressed only under certain conditions may escape detection when wild-type cells are grown under standard conditions. Here, we have taken a complementary approach where candidate outer membrane proteins have been identified by bioinformatics prediction, cloned and overexpressed, and finally localized by cell fractionation experiments. Out of eight predicted outer membrane proteins, we have confirmed the outer membrane localization for five-YftM, YaiO, YfaZ, CsgF, and YliI--and also provide preliminary data indicating that a sixth--YfaL--may be an outer membrane autotransporter.     

鲍曼不动杆菌外膜蛋白与耐药性分析

目的:分析35株鲍曼不动杆菌外膜蛋白(OMP)与耐药性的关系.方法:采用超声物理法制备鲍曼不动杆菌外膜蛋白标本,用变性聚丙烯酰胺凝胶电泳(SDSPAGE)检测外膜蛋白.直接荧光法测鲍曼不动杆菌对环丙沙星的吸收和积累.结果:35株鲍曼不动杆菌都有10条主要OMP带,耐药菌株与敏感菌株相比,发现外膜蛋白在约29 Ku条带处消失,而在26 Ku条带处却明显增强.耐药菌株药物积累量不及敏感菌株,经叠氮钠处理后,积累量上升并接近敏感菌株.结论:鲍曼不动杆菌耐药与外膜蛋白的低通透性和主动外运有关.