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PubMed2003

Restored functional immunogenicity of purified meningococcal PorA by incorporation into liposomes.

The impact of the conformation, lipooligosaccharide (LOS)-depletion and the presentation form of outer membrane protein PorA from Neisseria meningitidis (PorA) subtype P1.7-2,4 on the immune response in mice was studied. Native PorA was purified from outer membrane vesicles (OMVs) derived from meningococci and reconstituted into liposomes. The conformation of PorA after purification from OMVs and reconstitution in liposomes was monitored by use of electrophoretic and spectroscopic techniques and compared with the conformation of PorA in outer membrane complexes (OMCs) and heat-denatured PorA. The antigenicity of the PorA formulations was measured by ELISA by using a bactericidal anti-P1.4 monoclonal antibody. Immunogenicity was determined in Balb/c mice. PorA-specific IgG, isotype distribution and bactericidal activity were measured after subcutaneous immunization. In all formulations except in heat-denatured OMVs, PorA was present as trimers. The lipooligosaccharide (LOS) content was reduced by 96% in the purified protein with respect to the original OMVs. The antigenicity of purified PorA (i.e. ELISA response) was substantially higher as compared to PorA in liposomes, OMVs or OMCs. The results of the immunogenicity studies showed that all formulations were able to induce comparable IgG titers. However, whereas the antibodies raised by OMVs were bactericidal, the antibodies elicited by immunization with purified PorA were unable to kill meningococci. Remarkably, the ability to induce bactericidal antibodies was fully recovered by incorporation of the purified PorA into liposomes, in the absence of other adjuvants, as compared to LOS-containing OMVs.

Animals↗

PubMed1992

Review of chemosystematics: multivariate approaches to oral bacteria and yeasts.

There are several problems related to the classification and identification of bacterial and yeast species assigned to the genera Actinobacillus, Haemophilus, Pasteurella, Bacteroides, Prevotella, Porphyromonas, Campylobacter, Wolinella, Treponema, Candida, Torulopsis, and Saccharomyces, most of which belong to the resident oral microflora. The present review was written to demonstrate how multivariate analyses of data on cellular fatty acids, sugars, enzyme activities, and lysis kinetics during ethylenediaminetetraacetic acid (EDTA) and EDTA plus lysozyme treatment can be used to distinguish closely related species of these bacterial and yeast genera. With the exception of the Actinobacillus-Haemophilus-Pasteurella group, fatty acids were more discriminating than sugars. Enzymes from whole cells and outer membrane vesicles also contributed to taxonomic distinction. Apparently, chemosystematics, involving multivariate analyses, is a useful adjunct in oral microbial taxonomy.

Actinobacillus↗

PubMed2002

Extracellular pH modulates Helicobacter pylori-induced vacuolation and VacA toxin internalization in human gastric epithelial cells.

In this study we investigated whether an acidic extracellular pH may inhibit H. pylori-induced internalization of bacterial virulence factors by gastric epithelium, thus preventing ingestion of potentially dangerous luminal contents and resulting cellular damage. The interaction of H. pylori VacA toxin and ammonia (produced by H. pylori urease) with partly polarized gastric MKN 28 cells in culture was investigated at neutral and moderately acidic pH (6.2, compatible with cell viability) by means of neutral red dye uptake and ultrastructural immunocytochemistry. We found that acidic extracellular pH virtually abolished both VacA-dependent and ammonia-dependent cell vacuolation, as shown by the neutral red test, and caused a 50% decrease in VacA internalization into endosomal vesicles and vacuoles, as assessed by quantitation of immunogold particles. In addition, acidic pH blocked endosomal internalization of H. pylori outer membrane vesicles, a convenient indicator of endocytosis. Our data raise the possibility that suppression of gastric acid may increase H. pylori-induced gastric damage by enhancing epithelial internalization of H. pylori virulence factors through activation of endocytosis. Increased transmembrane diffusion of ammonia could also contribute to this process.

Bacterial Proteins↗

PubMed1987

Release of pertussis toxin and its interaction with outer-membrane antigens.

The absence of subunit S3 in cell-associated pertussis toxin (PT) from a mutant of Bordetella pertussis which failed to produce cell-free toxin suggested that this subunit was involved in the release of PT into the culture medium. The addition of methylated beta-cyclodextrin (MCD) to the culture medium caused a small but consistent increase in the release of lipopolysaccharide (LPS) by four wild-type strains of B. pertussis. Since previous studies have shown that MCD also enhances the levels of PT in culture supernates, it seemed probable that the increased shedding of outer-membranes vesicles (OMV) may explain the increased levels of both cell-free PT and LPS. Release of PT was inhibited in media buffered with HEPES but was unaffected in Tris/HC1 buffer. This suggested that in addition to shedding of the outer membrane, increased permeability and greater destabilization of the outer membrane, as caused by Tris/HC1 buffer, may be important in the release of PT. Our data do not support the idea that PT is packaged into OMV because only an insignificant proportion (0.01%) of the total cell-free PT was associated with LPS. The association of PT with small micelles derived from outer-membrane amphiphiles may be more important since the LPS content of PT purified from culture supernates (containing no large OMV) was nearly 18% by weight.

Antigens, Bacterial↗

PubMed2006

Borrelia burgdorferi BBA74, a periplasmic protein associated with the outer membrane, lacks porin-like properties.

The outer membrane of Borrelia burgdorferi, the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a Borrelia burgdorferi plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% alpha-helix with little beta-sheet structure. BBA74 in intact B. burgdorferi cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins.

Bacterial Outer Membrane Proteins↗

PubMed2006

External membrane vesicles from Helicobacter pylori induce apoptosis in gastric epithelial cells.

The Helicobacter pylori infection of gastric mucosa is one of the most common infectious diseases and is associated with a variety of clinical outcomes, including peptic ulcer disease and gastric cancer. Helicobacter pylori-induced damage to gastric mucosal cells is controlled by bacterial virulence factors, which include VacA and CagA. Outer membrane vesicles are constantly shed by the bacteria and can provide an additional mechanism for pathogenicity by releasing non-secretable factors which can then interact with epithelial cells. The present report shows that external membrane vesicles are able to induce apoptosis not mediated by mitochondrial pathway in gastric (AGS) epithelial cells, as demonstrated by the lack of cytochrome c release with an activation of caspase 8 and 3. Apoptosis induced by these vesicles does not require a classic VacA+ phenotype, as a negative strain with a truncated and therefore non-secretable form of this protein can also induce cell death. These results should be taken into account in future studies of H. pylori pathogenicity in strains apparently VacA-.

Apoptosis↗

PubMed1995

Actin-dependent mitochondrial motility in mitotic yeast and cell-free systems: identification of a motor activity on the mitochondrial surface.

Using fluorescent membrane potential sensing dyes to stain budding yeast, mitochondria are resolved as tubular organelles aligned in radial arrays that converge at the bud neck. Time-lapse fluorescence microscopy reveals region-specific, directed mitochondrial movement during polarized yeast cell growth and mitotic cell division. Mitochondria in the central region of the mother cell move linearly towards the bud, traverse the bud neck, and progress towards the bud tip at an average velocity of 49 +/- 21 nm/sec. In contrast, mitochondria in the peripheral region of the mother cell and at the bud tip display significantly less movement. Yeast strains containing temperature sensitive lethal mutations in the actin gene show abnormal mitochondrial distribution. No mitochondrial movement is evident in these mutants after short-term shift to semi-permissive temperatures. Thus, the actin cytoskeleton is important for normal mitochondrial movement during inheritance. To determine the possible role of known myosin genes in yeast mitochondrial motility, we investigated mitochondrial inheritance in myo1, myo2, myo3 and myo4 single mutants and in a myo2, myo4 double mutant. Mitochondrial spatial arrangement and motility are not significantly affected by these mutations. We used a microfilament sliding assay to examine motor activity on isolated yeast mitochondria. Rhodamine-phalloidin labeled yeast actin filaments bind to immobilized yeast mitochondria, as well as unilamellar, right-side-out, sealed mitochondrial outer membrane vesicles. In the presence of low levels of ATP (0.1-100 microM), we observed F-actin sliding on immobilized yeast mitochondria. In the presence of high levels of ATP (500 microM-2 mM), bound filaments are released from mitochondria and mitochondrial outer membranes. The maximum velocity of mitochondria-driven microfilament sliding (23 +/- 11 nm/sec) is similar to that of mitochondrial movement in living cells. This motor activity requires hydrolysis of ATP, does not require cytosolic extracts, is sensitive to protease treatment, and displays an ATP concentration dependence similar to that of members of the myosin family of actin-based motors. This is the first demonstration of an actin-based motor activity in a defined organelle population.

Actin Cytoskeleton↗

PubMed1995

Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease.

Animals↗

PubMed2005

Lectin-binding characteristics of a Lyme borreliosis spirochete Borrelia burgdorferi sensu stricto.

Borrelial glycoconjugates were localized by labeled lectins on ultrathin cryosections and on surfaces of intact negatively stained bacteria. Protein-saccharide complexes in these glycoconjugates were partially characterized by means of enzyme deglycosylation and mild alkali pretreatment of cryosections. The results of labeling were examined by transmission electron microscopy. Statistically evaluated results (relative labeling index, chi2 test) of gold labeling indicated that surfaces of Borrelia burgdorferi strain B31 and external (outer) membrane vesicles (MVs) were covered with glycoconjugates containing O-glycosidically linked N-acetyl-D-galactosamine (GalNAc) and N-glycosidically linked N-acetyl-D-glucosamine (GlcNAc). The presence of N-linked GalNAc, sialic acid, mannose and fucose on the surfaces of outer membranes and MVs was probably due to an adherence of BSK-H medium components, especially rabbit serum, to Borrelia surfaces.

Animals↗

PubMed2003

Relative immunogenicity of PorA subtypes in a multivalent Neisseria meningitidis vaccine is not dependent on presentation form.

The hexavalent meningococcal vaccine HexaMen, containing six PorAs on two vesicles, was tested in clinical studies. Although fourfold increases in serum bactericidal activity (SBA) titers against all of the PorAs were observed, there were significant differences between PorA-specific SBA titers. SBA titers were mainly directed against one PorA from each vesicle, P1.5-2,10 and P1.5-1,2-2, and were lower against the other PorAs, especially P1.7-2,4 and P1.19,15-1. We investigated whether these differences were due to immunological interference that resulted in competition between the three PorAs on the same vesicle or whether they were caused by a difference in the immunogenicities of the separate PorAs. Therefore, mice were immunized either with HexaMen, with six monovalent outer membrane vesicles (OMVs) representing the same six PorAs simultaneously (HexaMix), or with only one of the monovalent OMVs. The immunoglobulin G and SBA titers after HexaMen immunization in mice resembled the results obtained in clinical studies. Although immunization with HexaMix gave higher titers than immunization with HexaMen for some PorAs, the pattern of high and low titers was the same. Similar differences in immunogenicity between subtypes were seen after monovalent immunization when interference was eliminated as a cause of the differences. Monovalent immunization resulted in higher titers for P1.5-1,2-2 and P1.7,16 than immunization with HexaMen. However, no significant differences were found for the weakly immunogenic PorAs, P1.7-2,4 and P1.19,15-1. Since immunization with the six PorAs in the trivalent presentation form (HexaMen) and in the mixture of monovalent vesicles (HexaMix) resulted in the same pattern of high and low titers, we concluded that the differences between the PorA-specific responses are due to differences in the immunogenicities of the various PorAs and not due to interference that results in competition between different PorAs.

Animals↗

PubMed2001

Evaluation of cross-reactive antigens as determinants of cross-bactericidal activity in pathogenic and commensal Neisseria.

Several antisera raised against outer membane vesicles obtained from invasive and carrier Neisseria meningitidis strains and commensal Neisseria and Moraxella catharralis species were assayed to test cross-bactericidal activity on Neisseria meningitidis strains. Results demonstrate that, despite the wide antigenic cross-reactivity previously shown by Western-blotting for the major outer membrane antigenic proteins of all Neisseria species, complement mediated killing shows very variable patterns that can not be predicted on the basis of antigenic cross-reactivity. Results of antibody tritations on homologous and heterologous strains, isotyping, and bactericidal activity of sera raised against denatured purified outer-membrane vesicle proteins, suggest that the responsibility for most of the bactericidal activity of the sera must be conformational and/or shared epitopes not detectable by Western-blotting.

Animals↗

PubMedDate not supplied

Humoral immune response in hens naturally infected with Salmonella Enteritidis against outer membrane proteins and other surface structural antigens.

A simple procedure for obtaining surface exposed antigens of Salmonella Enteritidis is described. A heat treatment of whole bacteria in saline solution induced the release of small membrane vesicles containing outer membrane components as well as surface appendage components, such as fimbriae and flagellin. The characterization of the structural components of this extract, called HE, was established by SDS-PAGE and immunoblotting using polyclonal and monoclonal specific antibodies. Five major groups of proteins were identified: flagellin, porins, OmpA, SEF21 and SEF14 fimbriae. The immunogenicity of these proteins was studied by immunoblotting with serum samples from naturally infected hens. Flagellin, porins, OmpA, SEF14 and SEF21 fimbriae were immunogenic in the S. Enteritidis infected hens (frequency of reactants: 47.3, 97.3, 64.7, 50.0 and 60.8%, respectively); porins also reacted with sera from non infected hens (66.7%). The immunogenicity of these antigens in infected birds provide promise that they may serve as components of an effective subcellular vaccine for poultry salmonellosis.

Animals↗

PubMed1995

Human immune response against outer membrane proteins of Moraxella (Branhamella) catarrhalis determined by immunoblotting and enzyme immunoassay.

The role of Moraxella (Branhamella) catarrhalis as a respiratory tract pathogen is increasingly recognized. We looked at the human immune response against individual outer membrane proteins of M. catarrhalis and against the 81-kDa CopB protein, which has previously been shown to be a target for protective antibodies. Paired serum samples from six elderly patients with pneumonia were tested by Western blot (immunoblot) analysis by using outer membrane vesicles of M. catarrhalis 035E as antigen. All of the six convalescent-phase serum samples reacted with a protein which migrated at the position of the CopB protein and with a high-molecular-weight protein of M. catarrhalis; three serum samples also reacted with a 34-kDa outer membrane protein. Paired serum samples from 18 patients, 10 of which had M. catarrhalis infection on the basis of previous serology results, were tested by enzyme immunoassay (EIA) with the CopB protein and whole cells of M. catarrhalis 035E as antigens. Nine patients showed a significant rise in EIA titer between acute- and convalescent-phase sera when whole bacterial cells were used as antigens. Six (67%) patient samples that were positive by the EIA with the whole-cell antigen were also positive by the EIA with the CopB antigen, and six of nine patient samples negative by the EIA with the whole-cell antigen were also negative by the EIA with the CopB antigen. These results suggest that both the CopB and a high-molecular-weight protein are major targets of the immune response against M. catarrhalis, and further studies with greater amounts of patient materials are needed to elucidate the usefulness of CopB as an antigen in etiologic studies.

Aged↗

PubMed2000

Immune response to an 18-kilodalton outer membrane antigen identifies lipoprotein 20 as a Helicobacter pylori vaccine candidate.

Experiments were performed using the standardized murine model of Helicobacter pylori infection to determine the immunogenicity of H. pylori outer membrane vesicles in immune protection. These vesicles, which are naturally shed from the surface of the bacterium, induce a protective response when administered intragastrically to mice in the presence of cholera holotoxin, despite the absence of the urease enzyme and associated Hsp54 chaperonin. Immunoblotting identified a specific serum immunoglobulin G (IgG) response to an 18-kDa outer membrane protein in a significant number of immunized animals. This commonly expressed, immunodominant protein was subsequently identified as lipoprotein 20 (Lpp20). Hybridoma backpacks secreting an IgG1 subclass monoclonal antibody to Lpp20 were generated in H. pylori-infected mice and were found to significantly reduce bacterial numbers, providing evidence that this surface-exposed antigen is a true vaccine candidate and not merely an antigenic marker for successful, protective immunization.

Animals↗

PubMed2004

Meningococcal vaccines.

Meningococcal disease is one of the most feared and serious infections in the young and its prevention by vaccination is an important goal. The high degree of antigenic variability of the organism makes the meningococcus a challenging target for vaccine prevention. Meningococcal polysaccharide vaccines against serogroup A and C are efficacious and have been widely used, often in combination with serogroup Y and W135 components. Their relative lack of immunogenicity in young children and infants can be overcome by conjugation to a protein carrier. The effectiveness of serogroup C glycoconjugate vaccines in children of all ages has been demonstrated and they have now been introduced into routine vaccination schedules. Conjugate vaccines against other serogroups, including A, Y, and W135 will soon be available and it is hoped they may emulate this success. Prevention of serogroup B disease has proven more elusive. Several serogroup B vaccines based on outer membrane vesicles have been shown to be immunogenic and reasonably effective in adults and older children, but the protection offered by them is chiefly strain-specific. Multivalent recombinant PorA vaccines have been developed to broaden the protective effect, but no efficacy data are available as yet. Intensive efforts have been directed at other outer membrane protein vaccine candidates and lipopolysaccharide, and some of these have been shown to offer protection in experimental animal models. Nonpathogenic Neisseriae spp. such as Neisseria lactamica are also possible vaccine candidates. Previously unknown proteins have been identified from in silico analysis of the meningococcal genome and their vaccine potential explored. However, none of these has yet been presented as the 'universal' protective antigen and work in this field continues to be held back by our limited knowledge concerning the mechanisms of natural protection against serogroup B meningococci.

Adolescent↗

PubMed1987

cAMP-dependent protein kinase isozymes with preference for histone H2B as substrate in mitochondria of bovine heart.

Mitochondria from bovine hearts were fractionated by three different procedures and the fractions were characterized by marker enzymes. Highly purified outer membranes, membrane vesicles, and inner membranes, as well as two high-speed soluble fractions, were obtained. Azide (or oligomycin) resistant ATPase was not found to be a marker for outer membranes. The data were consistent with the association of the protein kinase activity with the soluble matrix of the mitochondria. Activity was highest with histone H2B as the substrate, with histone H1 next in preference. In contrast to the mitochondrial protein kinases studied previously, protamine, casein, and phosvitin were very poor substrates and there was no detectable phosphorylation of pyruvate dehydrogenase. Activity was stimulated by cAMP but not by cGMP, calmodulin, or phosphatidylserine--diolein, with or without Ca2+. Two cAMP-dependent isozymes were separated from the soluble fraction of the mitochondria by chromatography on DE-52 columns. Phosphorylation of histone H2B by the isozymes was inhibited by 98% by Kemptide.

Animals↗

PubMed1990

Assembly of an in vitro synthesized Escherichia coli outer membrane porin into its stable trimeric configuration.

The folding of in vitro synthesized outer membrane protein PhoE of Escherichia coli was studied in immunoprecipitation experiments with monoclonal antibodies which recognize cell surface-exposed conformational epitopes. The signal sequence appears to interfere with the formation of these conformational epitopes, since a mutant PhoE protein which lacks the majority of the signal peptide could be precipitated four times better than the wild type precursor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitated PhoE protein revealed that part of the immunoprecipitated PhoE was present as a heat-modifiable form of the protein which migrated faster in the gels than the completely denatured protein. This form of the protein probably represents a folded monomer which might be an intermediate in the assembly of the protein. Outer membrane vesicles were required to induce the formation of small amounts of heat-stable trimers, the functional form of the protein in vivo.

Amino Acid Sequence↗

PubMed2006

Proteome analysis of mitochondrial outer membrane from Neurospora crassa.

The mitochondrial outer membrane mediates numerous interactions between the metabolic and genetic systems of mitochondria and the rest of the eukaryotic cell. We performed a proteomic study to discover novel functions of components of the mitochondrial outer membrane. Proteins of highly pure outer membrane vesicles (OMV) from Neurospora crassa were identified by a combination of LC-MS/MS of tryptic peptide digests and gel electrophoresis of solubilized OMV proteins, followed by their identification using MALDI-MS PMF. Among the 30 proteins found in at least three of four separate analyses were 23 proteins with known functions in the outer membrane. These included components of the import machinery (the TOM and TOB complexes), a pore-forming component (porin), and proteins that control fusion and fission of the organelle. In addition, proteins playing a role in various biosynthetic pathways, whose intracellular location had not been established previously, could be localized to the mitochondrial outer membrane. Thus, the proteome of the outer membrane can help in identifying new mitochondria-related functions.

DNA, Recombinant↗