Outer Membrane Proteins (outer + membrane_protein)

Distribution by Scientific Domains
Distribution within Life Sciences

Kinds of Outer Membrane Proteins

  • kda outer membrane protein
  • major outer membrane protein

  • Selected Abstracts

    Isolation and Characterization of a Porin-Like Outer Membrane Protein from Xanthomonas campestris pv. campestris

    IUBMB LIFE, Issue 1 2002
    Lingyun Wang
    Abstract Xanthomonas campestris pv. campestris, a plant-associated pathogenic bacterium, is the causal agent of foliar spots and blights in crucifers. The major outer membrane protein, Omp37, of 37 kDa, has been identified, purified to homogeneity, and its characterization has also been carried out. Native Omp37 behaved as a trimer, as revealed by gel filtration and SDS-PAGE. FTIR measurements revealed a high ,-structure content. The pore-forming ability of the purified Omp37 was studied by the liposome swelling assay. Omp37, to our knowledge, is the first porin that has been isolated from Xanthomonas . This study clearly demonstrates that Omp37 is related to the family of trimeric bacterial porins. [source]

    Relevance of incubation temperature for Vibrio salmonicida vaccine production

    D.J. Colquhoun
    Aims:,To investigate the relationships between water temperature, bacterial growth, virulence and antigen expression in Vibrio salmonicida, the causal agent of cold water vibriosis in Atlantic salmon (Salmo salar L.). Methods and Results:,The significance of sea temperature was investigated using historical clinical and oceanographic data. An upper threshold for disease of approx. 10C was established. The effects of culture temperature and media type on bacterial growth were studied on solid and in liquid media. The highest rates of cell division were identified at 15C on solid media and 10C in liquid media. Outer membrane protein (OMP) expression and serological response in Atlantic salmon were studied using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. A novel 76-kDa OMP produced in unshaken cultures at 10C was not found to stimulate a specific humoral response. Conclusions:,Diagnostic agar plate-based incubation of suspected V. salmonicida should be carried out at 15C. High yield broth cultures for vaccine production should be incubated at 10C or lower. Significance and Impact of the Study:,This study is, to the best of our knowledge, the first to identify different optimal temperatures in a bacterial species cultured on physically different types of media. The evidence presented suggests that V. salmonicida and possibly other bacteria destined for vaccine use in poikilothermic organisms should be cultured at temperatures consistent with that at which disease occurs. [source]

    Crystallization and preliminary crystallographic studies of the C-terminal domain of outer membrane protein A from enterohaemorrhagic Escherichia coli

    Jiang Gu
    Outer membrane protein A (OmpA) of enterohaemorrhagic Escherichia coli (EHEC) plays multiple roles in bacterial physiology and pathogenesis, such as mediation of bacterial conjunction, maintenance of cell shape, induction of adhesion of EHEC to host cells etc. Better understanding of the functions of OmpA will help in the control of EHEC infections. OmpA is composed of two domains: the N-terminal domain and the C-terminal domain. The N-terminal domain is a ,-barrel structure and embeds in the outer membrane of the bacterium. The structure and function of the C-terminal domain of OmpA (OmpAC) remain elusive. In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7, resolution. The crystals belonged to space group I4132, with unit-cell parameter a = 158.99,. The Matthews coefficient and solvent content were calculated to be 2.55,3,Da,1 and 51.77%, respectively, for two molecules in the asymmetric unit. [source]

    Identification and characterization of Xenopus OMP25

    Masafumi Inui
    This study describes the isolation of mitochondrial outer membrane protein 25 (OMP25) from Xenopus laevis and an analysis of its role in early development. X. laevis OMP25 (xOMP25) is a transmembrane protein of the mitochondrial outer membrane with a PDZ domain in the cytoplasmic tail, and an approximate molecular size of 25 kDa. We isolated xOMP25 from a cDNA library of X. laevis tailbud embryos. Amino acid sequence analysis of xOMP25 showed 57% identity to mouse OMP25, with 73% identity in the PDZ domains. XOMP25 mRNA is expressed maternally, and at a constant level throughout early development. The transcript is localized to eye, otic vesicle, branchial arch and neural tube. Mitochondrial targeting of an EGFP-fusion protein of xOMP25 was visualized using a mitochondria-specific fluorescent dye. Overexpression of xOMP25 in embryos caused curved axes, small eyes and disorganized head structures. Knockdown of xOMP25 protein using antisense morpholino oligonucleotides resulted in slightly shortened axes and decreased neural tissue. Although the mechanism remains unclear, our results implicate xOMP25 protein in the formation of the intact neural tube. [source]

    Comparison of three different PCR methods for detection of Brucella spp. in human blood samples

    E Navarro
    In the diagnosis of human brucellosis, PCR could be a more sensitive technique than blood cultures and more specific than conventional serological tests. We compared three different PCR methods for the detection of Brucella spp. and we studied whether human genomic DNA affect the sensitivity of three primer pairs for the detection of Brucella DNA in a peripheral-blood PCR assay. These three pairs of primers amplified three different fragments included in: (i) a gene encoding a 31-kDa Brucella abortus antigen (primers B4/B5), (ii) a sequence 16S rRNA of B. abortus (primers F4/R2), and (iii) a gene encoding an outer membrane protein (omp -2) (primers JPF/JPR). The three primers assayed showed a difference in sensitivity for detecting purified Brucella DNA, ranging between 8 fg and 20 pg. However, the sensitivity of the primers F4/R2 and B4/B5 was affected by the presence of human DNA while the primers JPF/JPR were not. Therefore, although the sensitivity of PCR using primers F4/R2 is affected by human DNA, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis. [source]

    The role of Sov protein in the secretion of gingipain protease virulence factors of Porphyromonas gingivalis

    Keitarou Saiki
    Abstract Porphyromonas gingivalis transports Arg-gingipains and Lys-gingipain across the outer membrane via an unknown pathway. Recently, we found that the sov gene of P. gingivalis W83 was required for this step. In the present study, we characterized the Sov protein. We constructed a P. gingivalis strain that expresses histidine-tagged Sov instead of Sov. Subcellular fractionations and a histidine-tag pulldown experiment showed that histidine-tagged Sov was present in an outer membrane fraction. Furthermore, antiserum raised against the terminal regions of Sov obstructed the secretion of Arg-gingipains from wild-type W83 cells. A deletion study showed that the region from Phe2495 to the C-terminus Gln2499 of Sov is essential for gingipain secretion. Anti-histidine-tag immunoglobulins interfered with the secretion of Arg-gingipains by P. gingivalis cells that expressed histidine-tagged Sov. In conclusion, we found that Sov is an outer membrane protein participating in the secretion of gingipains and that the C-terminal region of Sov is exposed to the extracellular milieu and involved in the modulation of Sov function. [source]

    The Hek outer membrane protein of Escherichia coli is an auto-aggregating adhesin and invasin

    Robert P. Fagan
    Abstract Escherichia coli is the principal gram-negative causative agent of sepsis and meningitis in neonates. The pathogenesis of meningitis due to E. coli K1 involves mucosal colonization, transcytosis of epithelial cells, survival in the blood stream and eventually invasion of the meninges. The latter two aspects have been well characterized at a molecular level in the last decade. Less is known about the early stages of pathogenesis, i.e. adhesion to and invasion of gastrointestinal cells. Here, the characterization of the Hek protein is reported, which is expressed by neonatal meningitic E. coli (NMEC) and is localized to the outer membrane. It is demonstrated that this protein can cause agglutination of red blood cells and can mediate autoaggregation. Escherichia coli expressing this protein can adhere to and invade epithelial cells. So far, this is the first outer membrane protein in NMEC to be directly implicated in epithelial cell invasion. [source]

    Disruption of the Wolbachia surface protein gene wspB by a transposable element in mosquitoes of the Culex pipiens complex (Diptera, Culicidae)

    Y. O. Sanogo
    Abstract Culex pipiens quinquefasciatus Say and Culex pipiens pipiens Linnaeus are sibling species incriminated as important vectors of emerging and re-emerging infectious diseases worldwide. The two forms differ little morphologically and are differentiated mainly based upon ecological, behavioural, physiological and genetic traits. Within the North American zone of sympatry, populations of Cx. p. quinquefasciatus and Cx. p. pipiens undergo extensive introgression and hybrid forms have been reported in nature. Both Cx. p. quinquefasciatus and Cx. p. pipiens are infected with the endosymbiotic bacteria Wolbachia pipientis. Here, we report the presence of a transposable element belonging to the IS256 family (IS256wPip) associated with Wolbachia in both Cx. p. quinquefasciatus and Cx. p. pipiens populations. Using reverse transcriptase PCR and sequence analysis, we show that IS256wPip has disrupted the wspB locus, a paralogue of the Wolbachia outer membrane protein (wspA) gene. The inactivation of the wspB appears to be specific to Cx. p. quinquefasciatus and to hybrids of the two forms, and was not observed in the surveyed Cx. p. pipiens mosquitoes. Our results support the hypothesis of a different origin of North American Cx. p. quinquefasciatus and Cx. p. pipiens populations. The flux of mobile genetic elements in the Wolbachia wPip genome could explain the high level of crossing types observed among different Culex populations. The insertion of IS256wPip into wspB may comprise a genetic candidate for discriminating Wolbachia symbionts in Culex. [source]

    Lower antibody response to Porphyromonas gingivalis associated with immunoglobulin G Fc, receptor IIB polymorphism

    Y. Honma
    Background and Objective:, Human Fc,RIIB is one of the receptors for immunoglobulin G (IgG) and suppresses the activation of B lymphocytes through cross-linking with the B cell receptor via immune complexes. This function of Fc,RIIB is essential for the negative regulation of antibody production. Our previous study has demonstrated the gene polymorphism Fc,RIIB-I232T to be associated with periodontitis. The polymorphism Fc,RIIB-232T has been reported to inhibit B-cell antigen receptor signaling more effectively compared to Fc,RIIB-232I, while other groups concluded that Fc,RIIB-232T had no ability to inhibit activatory receptors. In this study, we examined whether Fc,RIIB-I232T polymorphism would change the IgG antibody response to the periodontopathic bacteria Porphyromonas gingivalis. Material and Methods:, Forty-seven patients with periodontitis were genotyped with the direct sequencing of genome DNA. Serum IgG and specific IgG subclass levels for the sonicate of P. gingivalis and the recombinant 40 kDa outer membrane protein (OMP) were determined. Results:, No significant difference in the total IgG level and IgG response to P. gingivalis sonicate were observed between sera from Fc,RIIB-232T carriers and non-carriers. The Fc,RIIB-232T carriers revealed a significantly lower IgG2 response to P. gingivalis 40 kDa OMP compared to non-carriers (p = 0.04, Mann,Whitney U -test). Lower responses of Fc,RIIB-232T carriers were also observed in specific IgG and IgG1 levels. The Fc,RIIB-232T carriers revealed a low level of IgG2 response to P. gingivalis 40 kDa OMP, even with a high average probing pocket depth. Conclusion:, These results suggest that association of the Fc,RIIB-232T allele with periodontitis might be related to the lower levels of antibody response to P. gingivalis. [source]

    Enhanced expression of vascular endothelial growth factor by periodontal pathogens in gingival fibroblasts

    Kanyarat Suthin
    Vascular endothelial growth factor (VEGF) has recently attracted attention as a potent inducer of vascular permeability and angiogenesis. Aberrant angiogenesis is often associated with lesion formation in chronic periodontitis. The aim of the present study was to investigate the properties of VEGF expression in human gingival fibroblasts (HGF) culture. HGF were stimulated with lipopolysaccharide (LPS), vesicle (Ve) and outer membrane protein (OMP) from Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. HGF constitutively produced VEGF and levels were significantly enhanced (P < 0.01) by stimulation with Ve and OMP from A. actinomycetemcomitans and P. gingivalis at concentrations of 10 g/ml or higher. On the other hand, VEGF levels were not increased by LPS stimulation. VEGF mRNA expression was also observed in Ve- and OMP-stimulated HGF. A vascular permeability enhancement (VPE) assay was performed using guinea pigs to ascertain whether supernatant from cultures of Ve- and OMP-stimulated HGF enhance vascular permeability in vivo. Supernatant from cultures of Ve- and OMP-stimulated HGF strongly induced VPE. This was markedly suppressed upon simultaneous injection of anti-VEGF polyclonal antibodies with the supernatant. Heating and protease treatment of the stimulants reduced the VEGF enhancing levels in Ve and OMP in vitro. These results suggest that Ve and OMP may be crucial heat-labile and protease-sensitive components of periodontal pathogens that enhance VEGF expression. In addition, VEGF might be associated with the etiology of periodontitis in its early stages according to neovascularization stimulated by periodontal pathogens causing swelling and edema. [source]

    Isolation of salt-sensitive mutants from Sinorhizobium meliloti and characterization of genes involved in salt tolerance

    W. Wei
    Abstract Aims:, The purpose of our research is to isolate salt-sensitive mutants and to study the genes involved in salt tolerance of the salt-tolerant bacterium Sinorhizobium meliloti 042BM. Methods:, Wild type S. meliloti 042BM bacteria are able to grow at a NaCl concentration of 0.6 mol l,1. A transposon Tn5-1063a mutagenesis library of S. meliloti 042BM was constructed and eight salt-sensitive mutants were isolated, which were unable to growth on FY plates containing 0.4 mol l,1 NaCl. Significance:, Our interest is to provide information about the mechanism of salt tolerance in bacteria by studying the genes involved in salt tolerance. Here, seven different genes were identified. These genes include omp10 encoding a cell outer membrane protein, relA encoding (p)ppGpp synthetase, greA encoding a transcription cleavage factor, nuoL encoding NADH dehydrogenase I chain L transmembrane protein, a putative nuclease/helicase gene and two unknown genes. Based on these findings, we suggest that the regulation of salt tolerance of S. meliloti 042BM is complex and on several levels. [source]

    Rapid detection of Haemophilus influenzae by hel gene polymerase chain reaction

    M.C. Yadav
    Abstract Aims: To evaluate the efficiency of hel gene polymerase chain reaction (PCR) to detect Haemophilus influenzae in various clinical/non-clinical samples. Methods and Results: Seventy-four clinical samples (cerebrospinal fluid, blood, sputum, throat and nasal swabs) and throat swabs of 17 asymptomatic carriers were collected. Primers were used to amplify the hel gene of H. influenzae encoding P4 outer membrane protein directly from the processed samples. The samples were also examined by conventional culture methods and the results were compared with those of PCR. The culture methods showed positive results in 60 (659%) of 91 samples in contrast to 62 (6812%) samples tested positive by PCR. None of the culture-positive samples were PCR-negative while two of the culture-negative samples were PCR-positive. The specificity of the products was confirmed by Southern hybridization and failure of various other organisms to amplify the hel gene product. The sensitivity of the PCR assay was found to be 50 pg of DNA. Conclusions: These findings suggest that the hel gene PCR is a rapid, sensitive and a specific new method for direct identification of H. influenzae. Significance and Impact of the study: Thus, this PCR test can improve the detection rate of H. influenzae in suspected clinical samples as compared with that of conventional culture methods. [source]

    The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium

    Alexandra Sittka
    Summary The RNA chaperone, Hfq, plays a diverse role in bacterial physiology beyond its original role as a host factor required for replication of Q, RNA bacteriophage. In this study, we show that Hfq is involved in the expression and secretion of virulence factors in the facultative intracellular pathogen, Salmonella typhimurium. A Salmonella hfq deletion strain is highly attenuated in mice after both oral and intraperitoneal infection, and shows a severe defect in invasion of epithelial cells and a growth defect in both epithelial cells and macrophages in vitro. Surprisingly, we find that these phenotypes are largely independent of the previously reported requirement of Hfq for expression of the stationary phase sigma factor, RpoS. Our results implicate Hfq as a key regulator of multiple aspects of virulence including regulation of motility and outer membrane protein (OmpD) expression in addition to invasion and intracellular growth. These pleiotropic effects are suggested to involve a network of regulatory small non-coding RNAs, placing Hfq at the centre of post-transcriptional regulation of virulence gene expression in Salmonella. In addition, the hfq mutation appears to cause a chronic activation of the RpoE-mediated envelope stress response which is likely due to a misregulation of membrane protein expression. [source]

    Identification of Xenorhabdus nematophila genes required for mutualistic colonization of Steinernema carpocapsae nematodes

    Kurt Heungens
    Summary One stage in the symbiotic interaction between the bacterium Xenorhabdus nematophila and its nematode host, Steinernema carpocapsae, involves the species-specific colonization of the nematode intestinal vesicle by the bacterium. To characterize the bacterial molecular determinants that are essential for vesicle colonization, we adapted and applied a signature-tagged mutagenesis (STM) screen to this system. We identified 15 out of 3000 transposon mutants of X. nematophila with at least a 15-fold reduction in average vesicle colonization. These 15 mutants harbour disruptions in nine separate loci. Three of these loci have predicted open reading frames (ORFs) with similarity to genes (rpoS, rpoE, lrp) encoding regulatory proteins; two have predicted ORFs with similarity to genes (aroA, serC) encoding amino acid biosynthetic enzymes; one, designated nilB (nematode intestine localization), has an ORF with similarity to a gene encoding a putative outer membrane protein (OmpU) in Neisseria; and three, nilA, nilC and nilD, have no apparent homologues in the public database. nilA, nilB and nilC are linked on a single 4 kb locus. nilB and nilC are > 104 -fold reduced in their ability to colonize the nematode vesicle and are predicted to encode membrane-localized proteins. The nilD locus contains an extensive repeat region and several small putative ORFs. Other than reduced colonization, the nilB, nilC and nilD mutants did not display alterations in any other phenotype tested, suggesting a specific role for these genes in allowing X. nematophila to associate with the nematode host. [source]

    Characterization and functional analysis of PorB, a Chlamydia porin and neutralizing target

    Aya Kubo
    A predicted protein (CT713) with weak sequence similarity to the major outer membrane protein (20.4% identity) in Chlamydia trachomatis was identified by Chlamydia genome analysis. We show that this protein is expressed, surface accessible, localized to the chlamydial outer membrane complex and functions as a porin. This protein, PorB, was highly conserved among different serovars, with nearly identical sequences between serovars D, B, C and L2. Sequence comparison between C. trachomatis and Chlamydia pneumoniae showed less conservation between species with 59.3% identity. Immunofluorescence staining with monospecific antisera to purified PorB revealed antigen localized within chlamydial inclusions and found throughout the developmental cycle. Antibodies to PorB neutralized infectivity of C. trachomatis in an in vitro neutralization assay confirming that PorB is surface exposed. As PorB was found to be in the outer membrane, as well as having weak structural characteristics similar to major outer membrane protein (MOMP) and other porins, a liposome-swelling assay was used to determine whether this protein had pore-forming capabilities. PorB had pore-forming activity and was shown to be different from MOMP porin activity. [source]

    Oral immunization with Porphyromonas gingivalis outer membrane protein and CpG oligodeoxynucleotides elicits T helper 1 and 2 cytokines for enhanced protective immunity

    C. Liu
    Summary The aim of this study was to evaluate the efficacy of an oral vaccine containing the 40-kDa outer membrane protein of Porphyromonas gingivalis (40K-OMP) and synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) to control oral infection by P. gingivalis. Oral immunization with 40K-OMP plus CpG ODN induced significant 40K-OMP-specific serum immunoglobulin G (IgG), IgA, and saliva IgA antibody responses. The 40K-OMP-specific CD4+ T cells induced by oral 40K-OMP plus CpG ODN produced both T helper type 1 (Th1; interferon-,) and Th2 (interleukin-4) cytokines. Furthermore, increased frequencies of CD11c+ B220+ dendritic cells (DCs) and CD11c+ CD11b+ DCs with upregulated expression of CD80, CD86, CD40, and major histocompatibility complex class II molecules were noted in spleen, Peyer's patches, and cervical lymph nodes. Immunized mice were then infected orally with P. gingivalis to determine whether the immune responses induced by oral 40K-OMP plus CpG ODN were capable of suppressing the bone resorption caused by P. gingivalis infection. Mice given 40K-OMP plus CpG ODN showed significantly reduced bone loss associated with oral infection by P. gingivalis. Oral administration of 40K-OMP together with CpG ODN induces Th1-type and Th2-type cells, which provide help for protective immunity against P. gingivalis infection. This may be an important tool for the prevention of chronic periodontitis. [source]

    Effects of Porphyromonas gingivalis antigens and proinflammatory cytokines on human coronary artery endothelial cells

    T. Honda
    Objective:, Individuals with periodontitis have been cited as having a significantly increased risk of developing coronary heart disease. Although accumulating evidence suggests that periodontal infection is involved in the development and progression of atherosclerosis, the underlying mechanisms remain to be elucidated. In the present study, we examined how periodontal infection could contribute to endothelial dysfunction. Methods:, Human coronary arterial endothelial cells were stimulated with tumor necrosis factor (TNF)-, and interleukin (IL)-1,, both of which are reported to be elevated in the serum of periodontitis patients. Cells were also stimulated with lipopolysaccharide, outer membrane protein and heat shock protein 60 derived from Porphyromonas gingivalis, a representative periodontopathic bacterium which is known to stimulate myeloid cells. Results:, Although TNF-, and IL-1,, at concentrations a little higher than those in sera of periodontitis patients, up-regulated the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, P. gingivalis antigens had only a slight stimulatory effect. Conclusion:, Experiments in which the total pathogen burden is considered, rather than a single species of bacteria, would increase our understanding of the contribution of which periodontal infection to atherogenesis. [source]

    Characterization and expression of a novel Porphyromonas gingivalis outer membrane protein, Omp28

    N. Slakeski
    We report the characterization of a Porphyromonas gingivalis gene, designated omp28, encoding a protein that we have previously purified and characterized as a 28-kDa outer membrane protein. The deduced amino acid sequence of the omp28 open reading frame displayed an outer membrane leader sequence and lipoprotein attachment site but did not exhibit any significant overall sequence identity with protein sequences in the databases. A small stretch of amino acids (19 residues) exhibits 50% sequence identity with a segment of a fimbrial protein from Dichelobacter nodosus involved in adhesion, suggesting that Omp28 may be a surface adhesin/receptor of P. gingivalis. Using the pET-24 vector we expressed recombinant Omp28 (rOmp28) in Escherichia coli. Western blot analyses of purified rOmp28 with rabbit antisera to a P. gingivalis outer membrane preparation, protective rat anti-whole P. gingivalis antisera and pooled human sera from chronic periodontitis patients showed that the recombinant was recognized by all antisera. Further, anti-rOmp28 antisera exhibited strong reactivity with a panel of four laboratory strains and 10 clinical isolates of P. gingivalis from the United States, Sudan, Romania and Norway. These results suggest that Omp28 is expressed by a wide distribution of P. gingivalis strains. [source]

    Identification and characterization of B-cell epitopes of a 53-kDa outer membrane protein from Porphyromonas gingivalis

    K. Oyaizu
    We have previously reported that Porphyromonas gingivalis FDC 381 possesses a 53-kDa protein antigen (Ag53) on its outer membrane that evokes a strong humoral immune response in many patients with periodontal disease and that the humoral immune responses to Ag53 differ greatly among patients. To understand how the individual humoral immune system against Ag53 was determined, the regions of Ag53 recognized by specific antibody (B-cell epitopes) and dominant subclasses of serum immunoglobulin G (IgG) against major B-cell epitopes were examined by enzyme-linked immunosorbent assay. This study used sera from six patients with periodontitis, which all reacted strongly with sonic extracts of P. gingivalis 381 and with purified Ag53, and sera from six periodontally healthy children, which did not react with either sonic extracts of P. gingivalis 381 or Ag53. The epitopes were identified using synthetic 5-residue overlapping decapeptides covering the entire Ag53. Thirteen of 89 synthetic decapeptides showed a strong reaction with sera from the periodontal patients, but no reaction with those from the healthy children. Four peptides of 13 exerted different immune responses among patients. Furthermore, restriction analyses of the highly antigenic regions revealed that three sequences, RAAIRAS, YYLQ and MSPARR, were identified as major B-cell epitopes. Additionally, these epitopes were recognized mainly by the IgG2 isotype. These data suggest that the difference of B-cell epitopes might influence individual differences in antibody titer against Ag53 and also that the epitopes recognized commonly by multiple antibodies are quite valuable for peptide vaccine development against P. gingivalis infection. [source]

    Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10

    Nicolai Juul
    Abstract The genome of the obligate intracellular bacteria Chlamydia pneumoniae contains 21 genes encoding polymorphic membrane proteins (Pmp). While no function has yet been attributed to the Pmps, they may be involved in an antigenic variation of the Chlamydia surface. It has previously been demonstrated that Pmp10 is differentially expressed in the C. pneumoniae CWL029 isolate. To evaluate whether the absence of Pmp10 in the outer membrane causes further changes to the C. pneumoniae protein profile, we subcloned the CWL029 isolate and selected a clone with minimal Pmp10 expression. Subsequently, we compared the proteome of the CWL029 isolate with the proteome of the subcloned strain and identified a specific cleavage of the C-terminal part of the major outer membrane protein (MOMP), which occurred only in the absence of Pmp10. In contrast, when Pmp10 was expressed we predominantly observed full-length MOMP. No other proteins appeared to be regulated according to the presence or absence of Pmp10. These results suggest a close association between MOMP and Pmp10, where Pmp10 may protect the C-terminal part of MOMP from proteolytic cleavage. [source]

    ORIGINAL ARTICLE: A Multi-Subunit Chlamydial Vaccine Induces Antibody and Cell-Mediated Immunity in Immunized Koalas (Phascolarctos cinereus): Comparison of Three Different Adjuvants

    Alison J. Carey
    Citation Carey AJ, Timms P, Rawlinson G, Brumm J, Nilsson K, Harris JM, Beagley KW. A multi-subunit chlamydial vaccine induces antibody and cell-mediated immunity in immunized koalas (Phascolarctos cinereus): comparison of three different adjuvants. Am J Reprod Immunol 2010; 63: 161,172 Problem, Chlamydial infections represent a major threat to the survival of the koala. Infections caused by Chlamydia pecorum cause blindness, infertility, pneumonia and urinary tract infections and represent a threat to the survival of the species. Little is known about the immune response in koalas, or the safety of commonly used adjuvants for induction of protective systemic and mucosal immunity. Method of study, In the present study, we immunized 18 healthy female koalas subcutaneously with a combination of three chlamydial antigens [major outer membrane protein (MOMP), NrdB and TC0512 (Omp85)] mixed with one of three different adjuvants [Alhydrogel, Immunostimulating Complex (ISC) and TiterMax Gold]. Results, All adjuvants induced strong neutralizing IgG responses in plasma against the three antigens with prolonged responses lasting more than 270 days seen in Alhydrogel and ISC immunized animals. Cloacal IgG responses lasting >270 days were also induced in ISC-immunized animals. Chlamydia -specific peripheral blood mononuclear cell proliferative responses were elicited by both Alhydrogel and ISC, and these lasted >270 days in the ISC group. Conclusion, The data show that a multi-subunit chlamydial vaccine, given subcutaneously, can elicit Chlamydia -specific cell-mediated and antibody responses in the koala demonstrating that the development of a protective vaccine is feasible. [source]

    Crystallization and preliminary crystallographic studies of MOMP (major outer membrane protein) from Campylobacter jejuni

    Jean Michel Bolla
    Campylobacter jejuni is the leading bacterial cause of human enteritis linked to ingestion of contaminated food or water. MOMP, the major outer membrane protein from these Gram-negative bacteria, belongs to the porin family. In order to determine the three-dimensional structure of this protein and to elucidate the underlying molecular mechanisms, the MOMP from C. jejuni strain 85H has been purified and crystallized by vapour diffusion. Two crystal forms were characterized for this membrane protein. X-ray diffraction data were collected to a resolution of 3.1, using a synchrotron-radiation source from the orthorhombic crystal form, which belonged to space group P21212 with unit-cell parameters a = 170.1, b = 101.9, c = 104.9,. With a trimer in the asymmetric unit, the solvent content is 64% (VM = 3.4,,Da,1). The other form exhibits trigonal symmetry (space group R3) with hexagonal unit-cell parameters a = b = 94.2, c = 161.2,, but diffracts X-rays poorly to about 4, with significant anisotropy. [source]

    Antibody responses to Porphyromonas gingivalis outer membrane protein in the first trimester

    Background:,Porphyromonas gingivalis (Pg) is one of the most harmful periodontal pathogens and it has been reported that Pg is associated with preterm birth (PTB), intrauterine growth retardation (IUGR) and pregnancy-induced hypertension (PIH), discovered by animal experiments and clinical research. The relationship between adverse pregnancy outcomes and maternal antibody response to Pg is controversial. On the other hand, the serum C-reactive protein (CRP) has been recognised as a reliable serum marker of periodontal disease. Aims:, To determine the significance of antibody responses to Pg affecting pregnancy outcomes in the first trimester. Methods:, A case,control study was carried out on women with PTB (n = 58), IUGR (n = 91), PIH (n = 32) and without any complications (control, n = 98). The serum level of the CRP and IgG1 against 40-kDa outer membrane protein of Pg (anti-40-kDa OMP Pg -IgG1) in the first trimester was measured. Results:, The IUGR group, and PTB patients whose placentas were diagnosed as chorioamnionitis or whose vaginal flora included Lactobacilli, showed a lower level of anti-40-kDa OMP Pg -IgG1 than the control group. There was no difference in the serum CRP level between each case group and control group. Conclusions:, These results suggest that a lack of humoral immunity against Pg in early pregnancy is associated with IUGR and some PTB. [source]

    Crystallization and preliminary crystallographic studies of the C-terminal domain of outer membrane protein A from enterohaemorrhagic Escherichia coli

    Jiang Gu
    Outer membrane protein A (OmpA) of enterohaemorrhagic Escherichia coli (EHEC) plays multiple roles in bacterial physiology and pathogenesis, such as mediation of bacterial conjunction, maintenance of cell shape, induction of adhesion of EHEC to host cells etc. Better understanding of the functions of OmpA will help in the control of EHEC infections. OmpA is composed of two domains: the N-terminal domain and the C-terminal domain. The N-terminal domain is a ,-barrel structure and embeds in the outer membrane of the bacterium. The structure and function of the C-terminal domain of OmpA (OmpAC) remain elusive. In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7, resolution. The crystals belonged to space group I4132, with unit-cell parameter a = 158.99,. The Matthews coefficient and solvent content were calculated to be 2.55,3,Da,1 and 51.77%, respectively, for two molecules in the asymmetric unit. [source]

    A multicopper oxidase is essential for manganese oxidation and laccase-like activity in Pedomicrobium sp.

    ACM 306
    Summary Pedomicrobium sp. ACM 3067 is a budding-hyphal bacterium belonging to the ,- Proteobacteria which is able to oxidize soluble Mn2+ to insoluble manganese oxide. A cosmid, from a whole-genome library, containing the putative genes responsible for manganese oxidation was identified and a primer-walking approach yielded 4350 bp of novel sequence. Analysis of this sequence showed the presence of a predicted three-gene operon, moxCBA. The moxA gene product showed homology to multicopper oxidases (MCOs) and contained the characteristic four copper-binding motifs (A, B, C and D) common to MCOs. An insertion mutation of moxA showed that this gene was essential for both manganese oxidation and laccase-like activity. The moxB gene product showed homology to a family of outer membrane proteins which are essential for Type I secretion in Gram-negative bacteria. moxBA has not been observed in other manganese-oxidizing bacteria but homologues were identified in the genomes of several bacteria including Sinorhizobium meliloti 1021 and Agrobacterium tumefaciens C58. These results suggest that moxBA and its homologues constitute a family of genes encoding an MCO and a predicted component of the Type I secretion system. [source]

    Immunisation with non-integral OMPs promotes pulmonary clearance of Pseudomonas aeruginosa

    Linda D. Thomas
    Abstract Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Lung damage due to chronic infections in cystic fibrosis sufferers is the major cause of morbidity and mortality in this group. The bacterium produces various immunomodulatory products that enable it to survive in the lung. Innate and increasing resistance to antibiotic therapy shown by this organism heightens the need for development of a vaccine. This study reports the identification of six non-integral protein antigens; Pa 13, azurin, acyl carrier protein (ACP), amidase, aminopeptidase and KatE, purified from a mucoid strain of P. aeruginosa. N-terminal amino acid sequencing was used to identify these proteins and, based on their ascribed functions, determined that their normal cellular location was cytosolic. A rat model of acute pulmonary infection was used to investigate the ability of these protein antigens to enhance pulmonary clearance of a live P. aeruginosa challenge. Mucosal immunisation with four of the six antigens significantly enhanced bacterial clearance from both the lavage fluid and lung tissue. The greatest level of clearance was demonstrated for the antigens; KatE, aminopeptidase and amidase. Enhanced bacterial clearance was maintained when the antigens amidase and aminopeptidase were produced in recombinant form. When delivered parenterally, aminopeptidase demonstrated its continued efficacy as a vaccine candidate. This study has demonstrated that non-integral outer membrane proteins are antigenic and protective and warrant further investigation as potential components of a vaccine. [source]

    Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins

    J. Rebire-Hut
    Abstract Bacterial adherence is a complex phenomenon involving specific interactions between receptors, including matricial fibronectin, and bacterial ligands. We show here that fibronectin and outer membrane proteins of Pseudomonas fluorescens were able to inhibit adherence of P. fluorescens to fibronectin-coated wells. We identified at least six fibronectin-binding proteins with molecular masses of 70, 55, 44, 37, 32 and 28 kDa. The presence of native (32 kDa) and heat-modified forms (37 kDa) of OprF was revealed by immuno-analysis and the 44-kDa band was composed of three proteins, their N-terminal sequences showing homologies with Pseudomonas aeruginosa porins (OprD, OprE1 and OprE3). [source]

    Pathogenesis of Helicobacter pylori Infection

    HELICOBACTER, Issue 2008
    Javier Torres
    Abstract The clinical outcome of Helicobacter pylori infection is determined by a complex scenario of interactions between the bacterium and the host. The main bacterial factors associated with colonization and pathogenicity comprise outer membrane proteins including BabA, SabA, OipA, AlpA/B, as well as the virulence factors CagA in the cag pathogenicity island (cagPAI) and the vacuolating cytotoxin VacA. The multitude of these proteins and allelic variation makes it extremely difficult to test the contribution of each individual factor. Much effort has been put into identifying the mechanism associated with H. pylori -associated carcinogenesis. Interaction between bacterial factors such as CagA and host signal transduction pathways seems to be critical for mediating the induction of membrane dynamics, actin-cytoskeletal rearrangements and the disruption of cell-to-cell junctions as well as proliferative, pro-inflammatory and antiapoptotic nuclear responses. An animal model using the Mongolian gerbil is a useful system to study the gastric pathology of H. pylori infection. [source]

    Pathogenesis of Helicobacter pylori Infection

    HELICOBACTER, Issue 2007
    Shin Maeda
    Abstract The clinical outcome of Helicobacter pylori infection is determined by a complex interaction between the bacterium and the host. The main bacterial factors associated with pathogenicity comprise outer membrane proteins, including BabA, SabA, OipA, AlpA, and AlpB, the vacuolating cytotoxin VacA and the products of cagPAI. The multitude of putative virulence factors makes it extremely difficult to test the contribution of each individual factor. Much effort has been put into identifying the mechanism associated with H. pylori -associated carcinogenesis. Interaction between bacterial factors such as CagA and host signal transduction pathways seems to be critical for mediating cell transformation, cell proliferation, invasion, apoptosis/anti-apoptosis, and angiogenesis. An animal model using the Mongolian gerbil is a useful model for showing gastric pathology due to H. pylori infection which is similar to that in humans and can be used to evaluate virulence factors including CagA, host responses, and environmental factors such as salt intake. [source]

    Pathogenesis of Helicobacter pylori Infection

    HELICOBACTER, Issue 2005
    Cu Figueiredo
    ABSTRACT As with many infectious diseases, only a fraction of people infected with Helicobacter pylori develop clinical disease, and host genetics, host immune response, and bacterial virulence factors appear to play critical roles. There has been considerable interest in putative bacterial virulence factors and, while several have been identified, it is not clear whether they act independently or in concert. Disease associations have been proposed for the cag pathogenicity island (PAI), vacA, and genes encoding outer membrane proteins (OMPs). Numerous studies published in the last year have provided new insights into the function of these putative virulence factors in gastroduodenal pathogenesis. This article will review the recent novel findings (from April 2004) for the roles of the putative disease-associated virulence factors as well as their interaction with host. [source]