Surface loop motion in FepA

Using a lysine-specific cleavable cross-linking reagent ethylene glycolbis(sulfosuccimidylsuccinate) (Sulfo-EGS), we studied conformational motion in the surface loops of Escherichia coli FepA during its transport of the siderophore ferric enterobactin. Site-directed mutagenesis determined that Sulfo-EGS reacted with two lysines, K332 and K483, and at least two other unidentified Lys residues in the surface loops of the outer membrane protein. The reagent cross-linked K483 in FepA L7 to either K332 in L5, forming a product that we designated band 1, or to the major outer membrane proteins OmpF, OmpC, and OmpA, forming band 2. Ferric enterobactin binding to FepA did not prevent modification of K483 by Sulfo-EGS but blocked its cross-linking to OmpF/C and OmpA and reduced its coupling to K332. These data show that the loops of FepA undergo conformational changes in vivo, with an approximate magnitude of 15 A, from a ligand-free open state to a ligand-bound closed state. The coupling of FepA L7 to OmpF, OmpC, or OmpA was TonB independent and was unaffected by the uncouplers CCCP (carbonyl cyanide m-chlorophenylhydrazone) and DNP (2,4-dinitrophenol) but completely inhibited by cyanide.     

Identification of immunogenic cell wall-associated proteins of Streptococcus suis serotype 2

Streptococcus suis serotype 2 (SS2) is a porcine and human pathogen with adhesive and invasive properties. The absence of suitable vaccine or virulent marker can be the bottleneck to control SS2 infection. An immunoproteome-based approach was developed to identify candidate antigens of SS2 for vaccine development. Hyperimmune sera, convalescent sera, and control sera were analyzed for reactivity by Western Blot against SS2 cell wall-associated proteins (WAPs) separated by 2-DE. A total of 34 proteins were identified by immunoproteomic analysis, of which 15 were recognized by both hyperimmune sera and convalescent sera, including most WAPs currently characterized as SS2 vaccine candidate antigens: muramidase-released protein (MRP), surface protein SP1 (Sao), and glyceraldehyde-3-phosphate dehydrogenase (GapdH). The novel immunogenic proteins may be developed as alternative antigens for further study of SS2 vaccine and diagnostics.     

Detecting the Dominant T and B Epitopes of Klebsiella pneumoniae Ferric Enterobactin Protein (FepA) and Introducing a Single Epitopic Peptide as Vaccine Candidate

International Journal of Peptide Research and Therapeutics - Klebsiella pneumoniae causes various human infections. Ferric enterobactin protein (FepA) is a conserved protein of K. pneumoniae with...     

Immunoproteomics of Helicobacter pylori infection and relation to gastric disease

The Gram negative bacterium Helicobacter pylori is a human pathogen which infects the gastric mucosa and causes an inflammatory process leading to gastritis, ulceration and cancer. A systematic, proteome based approach was chosen to detect candidate antigens of H. pylori for diagnosis, therapy and vaccine development and to investigate potential associations between specific immune responses and manifestations of disease. Sera from patients with active H. pylori infection (n = 24), a control group with unrelated gastric disorders (n = 12) and from patients with gastric cancer (n = 6) were collected and analyzed for the reactivity against proteins of the strain HP 26695 separated by two-dimensional electrophoresis. Overall, 310 antigenic protein species were recognized by H. pylori positive sera representing about 17% of all spots separated. Out of the 32 antigens most frequently recognized by H. pylori positive sera, nine were newly identified and 23 were confirmed from other studies. Three newly identified antigens which belong to the 150 most abundant protein species of H. pylori, were specifically recognized by H. pylori positive sera: the predicted coding region HP0231, serine protease HtrA (HP1019) and Cag3 (HP0522). Other antigens were recognized differently by sera from gastritis and ulcer patients, which may identify them as candidate indicators for clinical manifestations. The data from these immunoproteomic analyses are added to our public database (http://www.mpiib-berlin.mpg.de/2D-PAGE). This platform enables one to compile many protein profiles and to integrate data from other studies, an approach which will greatly assist the search for more immunogenic proteins for diagnostic assays and vaccine design.     

Identification of antigens from nosocomial <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> clinical isolates in sera from ICU staff and infected patients using the antigenome technique

Nosocomial infections with a bacterial origin are considered one of the most dangerous threats to global health. Among the causes of these infections, Acinetobacter baumannii is playing a significant role, and the present study aimed? to determine the immunogenic proteins of this bacteria. Clinical isolates of A. baumannii were obtained from positive sputum cultures of intensive care unit (ICU) patients confirmed by Polymerase chain reaction (PCR) of the OXA-51 gene, and sera was obtained from 20 colonized patients. In addition, 20 and 30 serum samples were collected from ICU nurses and healthy controls, respectively. All the samples were screened in the presence of antibodies against A. baumannii by enzyme-linked immunosorbent assay (ELISA). IgG purified from the serum samples by affinity chromatography was used to isolate the bacteria by the Magnetic-activated cell sorting (MACS) procedure. After the bacteria were cultured, the identified antigen proteins were studied by western blotting and Mass spectrometry (MS). The MS results were analyzed with MASCOT software and revealed a 35 KD protein, which corresponds to outer membrane protein A (OmpA) of A. baumannii, a 25 KD band, which is a carbapenem-associated resistance protein precursor, and a 60 KD protein band, identified as a stress-induced bacterial acidophilic repeat motif protein. According to the properties of immunogen antigens and bio informatics tools, the outer membrane proteins (OMPs) can be used as a vaccine candidate in animal models.     

Optimizing Expression of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> Surface Protein a,PspA: Serocross-Reactivity within Families of Antisera Induced Against Clades 1 and 3

Streptococcus pneumoniae is the agent responsible for infections such as pneumonia, otitis media, and meningitis. Among virulence factors, the Pneumococcal surface protein A (PspA) has been shown to be immunogenic and protective in mice, and is thus a good vaccine candidate. PspA has been classified into 6 clades and 3 families. Initially, pspA fragments, clades 1 and 3, were cloned into the pAE-6His expression vector. Proteins were expressed in Escherichia coli BL21(DE3) and purified by affinity and anion exchange chromatographies, with a yield of 11 mg/l of culture. Due to plasmid instability in E. coli, another construct using pspA1 was obtained based on pET-37b(+), which was shown to be stable in E. coli and increased the yield approximately 3-fold. Our results show good conditions for scale-up. Sera from immunized mice recognized PspA in total extracts of S. pneumoniae strains: anti-rPspA1p sera recognized native PspA clades 1 (+++), 2 (++) and 4 (+) and anti-rPspA3p sera recognized PspA clades 1 (+), 2 (+), 3 (+++) and 4 (+). The cross-reactivity pattern obtained confirms the notion that proteins from both families should be included for development of a broad-coverage vaccine; lower-cross reactivity between rPspAs of family 2 indicates that it may be necessary to include 2 proteins from this family.     

Immunoproteomic identification of 11 novel immunoreactive proteins of Riemerella anatipestifer serotype 2

Riemerella anatipestifer (RA) is one of the most important bacterial pathogens of ducks and other avian species worldwide. Twenty-one serotypes of RA have been identified, with RA serotype two (RA2) being reported as one of the most predominant serotypes underlying infections in China. Current approaches to the control of RA are hindered by the absence of effective vaccines, particularly those that exhibit cross-protection between different serotypes. In this study, a combination of two-dimensional electrophoresis, Western blot analysis and mass spectrometry were used to identify the antigenic proteins of RA2. A total of 16 immunoreactive proteins, representing 12 distinct proteins, were identified. These included OmpA, a known immunogenic protein of RA, as well as novel immunogens. PCR analysis also indicated that genes corresponding to each of the 12 distinct proteins were conserved among different RA serotypes. Eleven genes encoding these proteins were cloned and expressed in Escherichia coli BL21 (DE3). Eight of the 11 expressed proteins were able to react with hyperimmune rabbit serum against RAf153. One of these, recombinant elongation factor G, responded to RA2 sera but not RA1, whereas recombinant OmpA responded to both RA1 and RA2 sera. These data form a basis for the development of vaccine for both homologous and heterogeneous RA serotypes in addition to the production of target antigens for the development of diagnostic antibodies with the potential to distinguish between RA serotypes.     

Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells

Outer membrane protein A (OmpA) is a class of proteins highly conserved among the Enterobacteriaceae family and throughout evolution. Klebsiella pneumoniae is a capsulated gram-negative pathogen. It is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by a lack of an early inflammatory response. Data from our laboratory indicate that K. pneumoniae CPS helps to suppress the host inflammatory response. However, it is unknown whether K. pneumoniae employs additional factors to modulate host inflammatory responses. Here, we report that K. pneumoniae OmpA is important for immune evasion in vitro and in vivo. Infection of A549 and normal human bronchial cells with 52OmpA2, an ompA mutant, increased the levels of IL-8. 52145-Δwca(K2)ompA, which does not express CPS and ompA, induced the highest levels of IL-8. Both mutants could be complemented. In vivo, 52OmpA2 induced higher levels of tnfα, kc, and il6 than the wild type. ompA mutants activated NF-κB, and the phosphorylation of p38, p44/42, and JNK MAPKs and IL-8 induction was via NF-κB-dependent and p38- and p44/42-dependent pathways. 52OmpA2 engaged TLR2 and -4 to activate NF-κB, whereas 52145-Δwca(K2)ompA activated not only TLR2 and TLR4 but also NOD1. Finally, we demonstrate that the ompA mutant is attenuated in the pneumonia mouse model. The results of this study indicate that K. pneumoniae OmpA contributes to attenuate airway cell responses. This may facilitate pathogen survival in the hostile environment of the lung.     

An outer membrane protein, OmpK, is an effective vaccine candidate for Vibrio harveyi in Orange-spotted grouper (Epinephelus coioides)

The outer membrane proteins of the fish pathogen, Vibrio harveyi, have a role in interaction between bacterium and host and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein, OmpK, which serves as the receptor for broad-host-range vibriophage KVP40 in V. harveyi, was isolated and characterized. Then the OmpK gene coding for mature peptide was subcloned into prokaryotic expression vector pBV220 and transformed into Escherichia coli DH5 alpha strain. After temperature induction, a recombinant protein was detected about 28 kDa in molecular weight and accounted for 24.8% of total proteins of whole cell as estimated by SDS-PAGE and scanning analysis of gel image. Polyclonal antibodies were raised in rabbits against the purified protein and the reaction of the antibody was confirmed by western blotting using the purified protein and crude extract of V. harveyi. Orange-spotted groupers (Epinephelus coioides) vaccinated with recombinant OmpK produced specific antibodies, and were highly resistant to infection by virulent V. harveyi. These results indicate that the OmpK is an effective vaccine candidate against V. harveyi in Orange-spotted groupers.     

Immunogenic Burkholderia pseudomallei Outer Membrane Proteins as Potential Candidate Vaccine Targets

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. There is no vaccine towards the bacterium available in the market, and the efficacy of many of the bacterium''s surface and secreted proteins are currently being evaluated as vaccine candidates.

Methodology/Principal Findings

With the availability of the B. pseudomallei whole genome sequence, we undertook to identify genes encoding the known immunogenic outer membrane protein A (OmpA). Twelve OmpA domains were identified and ORFs containing these domains were fully annotated. Of the 12 ORFs, two of these OmpAs, Omp3 and Omp7, were successfully cloned, expressed as soluble protein and purified. Both proteins were recognised by antibodies in melioidosis patients'' sera by Western blot analysis. Purified soluble fractions of Omp3 and Omp7 were assessed for their ability to protect BALB/c mice against B. pseudomallei infection. Mice were immunised with either Omp3 or Omp7, subsequently challenged with 1×10⁶ colony forming units (cfu) of B. pseudomallei via the intraperitoneal route, and examined daily for 21 days post-challenge. This pilot study has demonstrated that whilst all control unimmunised mice died by day 9 post-challenge, two mice (out of 4) from both immunised groups survived beyond 21 days post-infection.

Conclusions/Significance

We have demonstrated that B. pseudomallei OmpA proteins are immunogenic in mice as well as melioidosis patients and should be further assessed as potential vaccine candidates against B. pseudomallei infection.     

Identification of vaccine candidate antigens of Staphylococcus aureus by serological proteome analysis

In this study we applied serological proteome analysis (Klade, C. S. et al. Proteomics 2001, 1, 890-898) for identification of bacterial vaccine candidate antigens. First, approximately one hundred sera from healthy individuals and patients suffering from Staphylococcus aureus infections were screened for antibodies against staphylococcal lysates and recombinant proteins representing surface antigens. Two pools (healthy donors, patients) each consisting of five sera with the highest antiproteinaceous IgG reactivity were selected. Second, S. aureus COL was grown under different conditions and the number of antigens expressed was monitored by Western blot analysis. Third, surface proteins were enriched by digesting the bacterial cell wall under isotonic conditions and subsequent removal of protoplasts. These protein preparations were resolved by two-dimensional electrophoresis (2-DE) (pI 4-7). 2-DE immunoblotting using the preselected serum pools at 1:10 000-1:100 000 dilutions revealed a number of highly immunogenic staphylococcal proteins. Twenty-one spots were isolated by preparative 2-DE, and analysed by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry sequencing of tryptic peptides. This led to the identification of 15 proteins including known and novel vaccine candidates. Seroreactivity of several antigens including serine-aspartate repeat containing protein D, immuno-dominant staphylococcal antigen and a novel 309 amino acid lipoprotein was independently confirmed by enzyme-linked immunosorbent assay and Western blot analysis of purified recombinant proteins. In conclusion, serological proteome analysis proved to be a powerful tool for the identification of novel staphylococcal antigens, which provide a basis for rational vaccine design.     

Streptococcus pneumoniae heat shock protein 70 does not induce human antibody responses during infection

Mouse monoclonal antibodies (mAbs) were developed against Streptococcus pneumoniae in search for potential common pneumococcal proteins as vaccine antigens. mAb 230,B-9 (IgG1) reacted by immunoblotting with a 70-kDa protein which was isolated by immunoaffinity chromatography and subsequent preparative electrophoresis. N-terminal amino acid sequencing showed homology to that of heat shock protein 70 (hsp70). The hsp70 epitope reactive with mAb 230,B-9 was found in all the pneumococci examined as well as in other streptococci and enterococci. The epitope was not expressed in several other examined Gram-positive or -negative bacteria. Pneumococcal hsp70 has by other investigators been proposed to be a vaccine candidate. Binding experiments using flow cytometry showed that the epitope was not surface-exposed on live exponential phase grown S. pneumoniae. Human patient sera did not react with affinity-purified pneumococcal hsp70. Therefore the pneumococcal hsp70 does not seem to be of special interest in a vaccine formulation. The human sera contained antibodies to high molecular proteins co-purified with hsp70. Some of these proteins could be the pneumococcal surface protein A.     

Identification of Omp38 by immunoproteomic analysis and evaluation as a potential vaccine antigen against Aeromonas hydrophila in Chinese breams

Aeromonas hydrophila is a fish pathogen causing systemic infections in aquatic environments, and determining its antigenic proteins is important for vaccine development to reduce economic losses in aquaculture worldwide. Here, an immunoproteomic approach was used to identify immunogenic outer membrane proteins (OMPs) of the Chinese vaccine strain J-1 using convalescent sera from Chinese breams. Seven unique immunogenic proteins were identified by two-dimensional (2-D) electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-TOF-MS). One protein of interest, Omp38, was expressed, and its immunogenicity and protective efficacy were evaluated in Chinese breams. The two groups of fish immunized with the inactivated vaccine and recombinant Omp38 protein showed significant serum IgM antibody levels after vaccination, compared with the fish injected with PBS buffer. In addition, the superoxide dismutase (SOD) activity, lysozyme (LSZ) activity and phagocytosis activity of head kidney lymphocytes of immunized groups were significantly higher than those of the control. The fish receiving inactivated vaccine and recombinant Omp38 protein developed a protective response to a live A. hydrophila challenge 45 days post-immunization, as demonstrated by increased survival of vaccinated fish over the control and by decreased histological alterations in vaccinated fish. Furthermore, protective effect was better in Omp38 group than in the inactivated vaccine group. These results suggest that the recombinant Omp38 protein could effectively stimulate both specific and non-specific immune responses and protect against A. hydrophila infection. Therefore, Omp38 may be developed as a potential vaccine candidate against A. hydrophila infection.     

Plasmodium vivax apical membrane antigen-1: comparative recognition of different domains by antibodies induced during natural human infection

The Apical Membrane Antigen-1 (AMA-1) of Plasmodium sp. has been suggested as a vaccine candidate against malaria. This protein seems to be involved in merozoite invasion and its extra-cellular portion contains three distinct domains: DI, DII, and DIII. Previously, we described that Plasmodium vivax AMA-1 (PvAMA-1) ectodomain is highly immunogenic in natural human infections. Here, we expressed each domain, separately or in combination (DI-II or DII-III), as bacterial recombinant proteins to map immunodominant epitopes within the PvAMA-1 ectodomain. IgG recognition was assessed by ELISA using sera of P. vivax-infected individuals collected from endemic regions of Brazil or antibodies raised in immunized mice. The frequencies of responders to recombinant proteins containing the DII were higher than the others and similar to the ones observed against the PvAMA-1 ectodomain. Moreover, ELISA inhibition assays using the PvAMA-1 ectodomain as substrate revealed the presence of many common epitopes within DI-II that are recognized by human immune antibodies. Finally, immunization of mice with the PvAMA-1 ectodomain induced high levels of antibodies predominantly to DI-II. Together, our results indicate that DII is particularly immunogenic during natural human infections, thus indicating that this region could be used as part of an experimental sub-unit vaccine to prevent vivax malaria.     

Differential immunogenicity and clinical relevance of kidney compartment specific antigens after renal transplantation

To evaluate the pathogenic role of non-HLA antibodies after organ transplantation, 81 unique serum samples from renal transplant patients were analyzed by protein array technology on integrative genomics approach (Li, L.; et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (11), 4148-53; Higgins, J. P.; et al. Mol. Biol. Cell 2004, 15 (2), 649-56), validated by ELISA, and the results correlated with clinical relevance with time post-transplantation or post-transplant graft function. There was a significant association of de novo non-HLA antibodies with time post-transplantation (n = 1,785) and decline in graft function over the subsequent year (n = 105). There was an enrichment of immunogenic antigens in the renal cortex (p = 0.01) with post-transplant time, and for glomerular specific targets (p = 0.02) with decline in graft function. Two targets with very strong correlation in each category (AGT and SPDYA) were validated by customized ELISA assays in independent patient sera and their localization confirmed by immunohistochemistry. In conclusion, defined profiles of these non-HLA antibodies to renal cortical proteins develop with increasing length of engraftment, and may reflect the increasing recognition of altered localization or exposure of renal tubular and interstitial proteins, affected by advancing chronic nonimmune graft injury. The panel of non-HLA antibodies to glomerular targets is most interesting, as these corresponding antigenic targets may be important pathways in functional graft injury and could provide novel targets for drug design.     

副溶血性弧菌外膜蛋白K的B细胞线性表位预测

目的预测副溶血性弧菌外膜蛋白K(OmpK)的B细胞线性表位。方法 NCBI下载已登录的OmpK的基因序列,对其进行生物信息学分析,应用DNAStar protean软件综合分析OmpK蛋白的二级结构、柔性、表面可能性、亲水性和抗原指数等多种参数,预测其B细胞线性表位。结果 OmpK蛋白的优势B细胞线性表位位于肽链的第7-13、25-36、63-69、140-147、182-188、234-239区段。结论预测得到OmpK蛋白的6个优势B细胞线性表位,为进而克隆表达串联表位蛋白,研制副溶血性弧菌多表位疫苗奠定基础。     

鸭疫里氏杆菌多种血清型间接ELISA检测方法的建立

【目的】鸭疫里氏杆菌(Riemerella anatipestifer,RA)是一种重要的禽病病原,分为21个血清型。但一直缺乏一种针对多种血清型广泛适用的抗体检测方法。前期的研究表明,外膜蛋白A (Outer membrane protein A,OmpA)广泛存在于多种血清型的RA菌株中,是一种重要的免疫原性蛋白,并且其基因序列在RA血清型之间具有高度的保守性,提示其可以作为RA感染血清抗体检测的靶点分子。以重组蛋白OmpA建立间接酶联免疫吸附试验检测RA的抗体。【方法】通过诱导表达条件的摸索及蛋白纯化,获得适用于ELISA包被的重组OmpA抗原。通过Western-blot证明重组蛋白OmpA是否与RA多种血清型发生免疫学反应。进行方阵试验以确定ELISA抗原的最佳包被浓度、被检测血清的反应浓度。重复性、特异性和敏感性试验检查该方法的实用性。【结果】实验证实加入1%乙醇的诱导培养基有利于重组蛋白的可溶性表达。Western-blot结果表明,重组蛋白OmpA可以与1、2、6、10、11、13、14和17型多种RA主要流行血清型有良好的免疫反应性。经方阵试验确定抗原的最佳包被浓度为8 mg/L,待检血清的最佳稀释度为1:160。所建立检测方法具有良好的重复性、特异性和敏感性。【结论】实验建立的鸭疫里氏杆菌多种血清型间接ELISA检测方法可以用于免疫后抗体消长以及感染性抗体的检测。     

A shared antigen among Vibrio species: Outer membrane protein-OmpK as a versatile Vibriosis vaccine candidate in Orange-spotted grouper (Epinephelus coioides)

The outer membrane protein-OmpK has been considered as a vaccine candidate for the prevention of infections due to Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus in fish. Interestingly, the polyclonal antibody raised against the recombinant OmpK from V. harveyi strain EcGs020802 recognized the OmpK homologues from other strains of Vibrio species by immunoblotting. The ompK genes from 19 Vibrio strains including V. harveyi (11), V. alginolyticus (6) and V. parahaemolyticus (2) were then cloned and sequenced. Alignment analysis based on the amino acid sequences indicated that the OmpK from V. harveyi strain EcGs020802 had 71.7–99.2% of identities with those from V. harveyi, V. alginolyticus and V. parahaemolyticus. Western blot analysis revealed that the corresponding native proteins ranged between 28 and 31 kDa, consistent with predicated molecular weight of OmpK in Vibrio strains. Furthermore, the cross-protective property of recombinant OmpK was evaluated through challenge with heterogeneous virulent Vibrio strains in Orange-spotted groupers (Epinephelus coioides). Orange-spotted groupers vaccinated with recombinant OmpK were more tolerant of the infection by virulent Vibrio strains and their relative percentage survival (RPS) was correlative with the degree of the identity of deduced amino acid sequences of their OmpK. Taken together, the OmpK is a conserved protective antigen among tested Vibrio species and might be a potentially versatile vaccine candidate for the prevention of infections due to V. harveyi, V. alginolyticus and V. parahaemolyticus.     

副溶血弧菌SH112株OmpA蛋白的高效表达及免疫学特性

【目的】我们前期研究表明副溶血弧菌SH112株的OmpA蛋白在该菌的致病过程中发挥重要作用,是亚单位疫苗研制的潜在靶标抗原。本研究进一步对ompA(VPA1186)基因进行克隆表达,并研究其免疫学特性。【方法】扩增去除信号肽序列的成熟外膜蛋白OmpA的基因片段,定向克隆至表达载体,基因测序后对其编码蛋白质进行生物信息学分析。重组蛋白His-OmpA经纯化后,免疫ICR小鼠制备鼠多抗血清。Western blotting检测该蛋白的免疫原性及鼠多抗血清的特异性。动物实验验证其免疫保护率。【结果】成功表达分子量约为40.0 kDa的重组蛋白His-OmpA。制备的鼠多抗血清ELISA效价可达1∶50000以上。Westernblotting检测结果显示,该血清可与His-OmpA蛋白、总外膜蛋白和全菌蛋白发生特异性反应,说明所表达的目的蛋白保持原蛋白的免疫原性。此外,该高免血清可与其他主要血清型的副溶血弧菌发生特异性交叉反应,而与其他非副溶血弧菌菌株无交叉反应,表明该血清特异性较高,且提示OmpA蛋白可能是副溶血弧菌属的共同保护性抗原。小鼠免疫保护实验结果表明,该蛋白可提供约35%的免疫保护率。【结论】OmpA蛋白可作为诊断副溶血弧菌感染和亚单位疫苗研制的靶蛋白,为进一步开展该蛋白的功能研究提供了参考。     

Demonstration of a bacteriophage receptor site on the Escherichia coli K12 outer-membrane protein OmpC by the use of a protease

The Escherichia coli K12 outer-membrane proteins OmpA, OmpC, OmpF, PhoE, and LamB (all of transmembrane nature) can serve as phage receptors. We have shown previously that one OmpA-specific phage, Ox2, can give rise to the host range mutants Ox2h10 and Ox2h12, with the latter being derived from the former [Morona, R. & Henning, U. (1984) J. Bacteriol. 159, 579-582]. Unlike Ox2, both host range phages can use the OmpA and OmpC proteins as receptors and Ox2h12 is better adapted to the OmpC protein than Ox2h10. In a search for the site(s) of OmpC protein involved in phage recognition, it was found that proteinase K is able to cleave all of the proteins mentioned above. OmpC protein (Mr = 38306) could be cleaved from outside the cell by proteinase K resulting in two fragments of Mr approximately equal to 21000 and Mr approximately equal to 17500. The use of OmpC-PhoE hybrid proteins allowed us to assign the approximately equal to 21000-Mr fragment to the CO2H-terminal moiety of the protein. Proteinase K treatment of intact cells abolished their activity to neutralize the OmpC-specific phage Tulb and reduced this ability towards phage Ox2h12. The OmpA, OmpF, PhoE and LamB proteins were cleaved by the protease not in intact cells but only when acting on cell envelopes. The sizes of the OmpC protein fragments and the results obtained with the hybrid proteins very strongly suggest that the protein is cleaved from outside the cell at a region involving amino acid residues 150-178 of the 346-residue protein, which shows homology to two regions of the OmpA protein which are involved in its phage receptor site (loc. cit.). These areas also exhibit some homology to a region of the LamB protein which is thought to be part of this protein's receptor site [Charbit et al. (1984) J. Mol. Biol. 175, 395-401]. This suggests that there is a common denominator for proteinaceous phage receptor site because the LamB-specific phage lambda and phage Tulb are of completely different nature. We conclude that the region of the OmpC protein in question is cell-surface-exposed and acts as a phage receptor site.