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PubMed1985

Association of resistance of Neisseria gonorrhoeae to killing by human phagocytes with outer-membrane proteins of about 20 kilodaltons.

The determinant(s) of gonococcal resistance to killing by human phagocytes has been extracted from outer membrane vesicles (OMV) of a phagocyte-resistant strain, BS4 (agar), with sodium cholate (1%, w/v). The extracts, like the OMV, nullified the effect of antiserum raised against whole BS4 (agar) to promote intracellular killing of the latter by human peripheral blood phagocytes. Fractionation of the extract on Sephadex G75 produced an active fraction with much less protein and lipopolysaccharide (LPS) than in the original extract. Furthermore, crude LPS prepared from the resistant gonococci was inactive. These results imply that the factor(s) promoting intracellular resistance is a protein. SDS-PAGE of the active fraction suggested that the factor was not a principal outer membrane protein nor one of three proteins previously thought to be associated with resistance. In contrast to a similar preparation from a phagocyte-susceptible strain, BSSH, the active fraction from BS4 (agar) showed faintly staining proteins in the regions of 20 and 60 kDal. When eluted from the gels, the former but not the latter neutralized the above effect of antisera, thus associating the 20 kDal protein(s) with resistance to intracellular killing.

Bacterial Outer Membrane Proteins↗
PubMed1991

Development of a second generation group B meningococcal vaccine.

Outer membrane protein vaccines have now been demonstrated to be effective in prevention of group B Neisseria meningitidis disease, but the extent and duration of protection needs improvement. To develop a second generation outer membrane protein vaccine we have begun studies to evaluate vaccines containing iron regulated proteins in lipopolysaccharide depleted outer membrane vesicles. Since the iron regulated proteins are critical for in vivo survival, it is our working hypothesis that antibodies to these proteins will either be bactericidal or block iron uptake. Four representative strains were selected, and preliminary studies indicate that vaccines containing the iron regulated proteins are immunogenic in mice.

Antibodies, Bacterial↗
PubMed2005

The role of matrix vesicles in growth plate development and biomineralization.

Skeletal cells control the initiation of mineralization in vivo and determine the selective distribution pattern of mineralization by releasing calcification-initiating, submicroscopic, extracellular matrix vesicles (MVs) at selected sites in the extracellular matrix. The overall objective of this review is to outline what is currently known about the mechanisms of MV biogenesis and mineral initiation, while emphasizing recent observations that enhance our understanding of these mechanisms. Data from studies on the general mechanism of biogenesis of outer membrane vesicles and the formation and function of non-skeletal matrix vesicles is presented to stimulate thought concerning the possible biological functions that these structures may share with MVs.

Alkaline Phosphatase↗
PubMed1997

T-Cell epitope mapping the PorB protein of serogroup B Neisseria meningitidis in B10 congenic strains of mice.

T-cell epitope mapping the meningococcal serotype 15 PorB protein performed in this study in three congenic strains of mice with B10 genetic background revealed at least three murine T-cell epitopes (55-72, 163-180, and 226-261), located in the highly conserved putative transmembrane regions of Neisserial porins. Proliferation assays with popliteal lymph node cells derived from mice immunized with the PorB protein or with synthetic 18-mer peptides showed that epitope 163-180 immunized only in the H-2d haplotype, epitope 55-72 could be presented by both H-2f and H-2s molecules, while the 226-261 region covered by three overlapping peptides could be efficiently recognized in context of all three MHC class II haplotypes studied. Inhibition experiments with blocking I-Aalpha- and I-Ealpha-specific mAb showed that peptide 163-180 was presented by I-Ad and peptide 244-261 was presented by both I-Af and I-As. In addition, evidence was obtained that peptide 226-243 was presented in context of H-2d or I-As haplotypes and peptide 55-72 was presented in context of I-Af and I-As loci. Finally, the Norwegian outer membrane vesicle vaccine, but not the purified PorB protein, could recall responses in mice immunized with synthetic peptides corresponding to the 226-261 region. Altogether, these results suggest that T-cell epitopes identified on the serotype 15 PorB protein, particularly those presented by several MHC class II molecules (e.g., 226-261), could have important implications for the development of meningococcal vaccines.

Amino Acid Sequence↗
PubMed1992

An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca.

The last gene (pulO) of the pulC-O pullulanase secretion gene operon of Klebsiella oxytoca codes for a protein that is 52% identical to the product of the pilD/xcpA gene required for extracellular protein secretion and type IV pilus biogenesis in Pseudomonas aeruginosa. The PilD/XcpA protein is known to remove the first six amino acids of the signal sequence of the type IV pilin precursor by cleaving after the glycine residue in the conserved sequence GF(M)XXXE (where X represents hydrophobic amino acids). This prepilin peptidase cleavage site is present in the products of four genes in the pulC-O operon (PulG, PulH, Pull and PulJ proteins). It is shown here that PulO processes the pulG gene product in vivo. Processing was maximal within 15 seconds, but experiments in which the expression of pulO was uncoupled from that of the other genes in the secretion operon suggest that processing can also occur post-translationally. The products of two pulG derivatives with internal inframe deletions were also processed by PulO, but the three PulG-PhoA hybrids, two PulJ-PhoA hybrids and the single PulH-PhoA hybrid tested did not appear to be processed. Sucrose gradient fraction experiments showed that both precursor and mature forms of PulG appear to be associated with low-density, outer membrane vesicles prepared by osmotic lysis of sphaeroplasts. Neither the xcpA gene nor the Bacillus subtilis gene comC, which is also homologous to pulO and codes for a protein with type IV prepilin peptidase activity, can correct the pullulanase secretion defect in an Escherichia coli strain carrying all of the genes required for secretion except pulO. Furthermore, neither XcpA nor ComC is able to process prePulG protein in vivo.

Amino Acid Sequence↗
PubMed2003

Recent developments in vaccines to prevent meningococcal serogroup B infections.

Meningococcal disease in most western countries is mainly caused by serogroup B. Despite the availability of successful meningococcal serogroup C conjugate vaccines, there is no effective vaccine against serogroup B. Efficacy trials with outer membrane vesicle (OMV) vaccines were ineffective and are complicated by the high variability of its main component, porin A. Several new approaches to either optimizing these OMV vaccines or searching for new, highly conserved antigens are therefore being investigated. The completion of the meningococcal genome sequence has provided new challenges. This review summarizes recent developments in the search for a broadly protective meningococcal serogroup B vaccine.

Animals↗
PubMed1996

Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete.

Amino Acid Sequence↗
PubMed1994

Humoral immune response to class 1 outer membrane protein during the course of meningococcal disease.

We have determined the amounts of specific anti-class 1 outer membrane protein antibodies in sera from 25 patients during the course of systemic meningococcal disease, using purified class 1 protein as the sensitizing antigen in an enzyme-linked immunosorbent assay. The class 1 protein was obtained from a variant of strain 44/76 (B:15:P1.7,16) lacking class 3 and class 4 outer membrane proteins. Specific anti-class 1 (serosubtype P1.7,16) outer membrane protein immunoglobulin G (IgG) antibody levels increased significantly in 12 patients (12 of 25; 48%), regardless of the serotype of the infecting strain, indicating that the antibodies reacted in part with epitopes not determined by the monoclonal antibodies used for serotyping. Most patients had low levels of anti-class 1 IgG antibodies during the acute illness. The antibody levels peaked during the second week of disease and returned to near baseline levels in sera collected 6 weeks to 12 months after the onset of the disease. The majority of the specific anti-class 1 IgG antibodies bound to surface-exposed epitopes on whole bacteria and belonged to the IgG1 and IgG3 subclasses. Anti-class 1 IgA and IgM antibodies were not detected in any of the patient sera. Prior to disease, seven patients had been immunized with a meningococcal outer membrane vesicle vaccine developed from strain 44/76 (P1.7,16). None of these patients was infected with meningococcal strains containing class 1 protein homologous or partly homologous to that of the vaccine strain, indicating serosubtype-specific protection. The highest anti-class 1 IgG antibody peak levels were seen in immunized patients infected with strains of heterologous serotype, suggesting an anamnestic response. However, these patients were not protected from meningococcal disease after immunization.

Adolescent↗
PubMed2001

Vaccine prevention of meningococcal disease, coming soon?

The past century has seen the use of a number of vaccines for prevention and control of meningococcal disease with varied success. The use of polysaccharide vaccines for the control of outbreaks of serogroup C infections in teenagers and young adults and epidemic serogroup A disease has been established for 30 years and an effective protein-polysaccharide conjugate vaccine against serogroup C was introduced into the UK infant immunisation schedule in 2000. The next generation of these glycoconjugate vaccines will be on the shelf soon, eventually offering the prospect of eradication of serogroups A, C, Y and W135 through routine infant immunisation. Despite these exciting prospects, serogroup B meningococci still account for a majority of infections in industrialised nations but development of safe, immunogenic and effective serogroup B meningococcal vaccines has been an elusive goal. Outer membrane vesicle vaccines for B disease are already used in some countries, and will likely be used more widely in the next few years, but efficacy for endemic disease in children has so far been disappointing. However, the innovations arising from the availability of the meningococcal genome sequence, public and scientific interest in the disease and recent pharmaceutical company investment in development of serogroup B vaccines may have started the countdown to the end of meningococcal infection in children.

Antigens, Bacterial↗
PubMed2006

Intranasal immunisation with inactivated RSV and bacterial adjuvants induces mucosal protection and abrogates eosinophilia upon challenge.

We have previously shown that following intranasal exposure to influenza virus, specific plasma cells are generated in the nasal-associated lymphoid tissue (NALT) and maintained for the life of the animal. However, we also showed that following infection with respiratory syncytial virus (RSV), specific plasma cells are generated in the NALT but wane quickly and are not maintained even after challenge, even though RSV-specific serum antibody responses remain robust. Only infection with influenza virus generated sterilising immunity, implying a role for these long-lived plasma cells in protection. We show here that the RSV-specific IgA NALT plasma cell population and lung antibody levels can be substantially boosted, both at acute and memory time points, by intranasal immunisation with inactivated RSV (iRSV) in combination with bacterial outer membrane vesicles (OMV) compared to live RSV alone. Finally, challenge with live RSV showed that immunisation with iRSV and OMV protect against both virus replication in the lung and the eosinophil infiltrate generated by either live RSV or iRSV alone. These data show that immunisation with iRSV and OMV maintains a NALT RSV-specific plasma cell population and generates an efficient protective immune response following RSV infection.

Adjuvants, Immunologic↗
PubMed1997

Sequence analysis and characterization of a 40-kilodalton Borrelia hermsii glycerophosphodiester phosphodiesterase homolog.

We report the purification, molecular cloning, and characterization of a 40-kDa glycerophosphodiester phosphodiesterase homolog from Borrelia hermsii. The 40-kDa protein was solubilized from whole organisms with 0.1% Triton X-100, phase partitioned into the Triton X-114 detergent phase, and purified by fast-performance liquid chromatography (FPLC). The gene encoding the 40-kDa protein was cloned from a B. hermsii chromosomal DNA lambda EXlox expression library and identified by using affinity antibodies generated against the purified native protein. The deduced amino acid sequence included a 20-amino-acid signal peptide encoding a putative leader peptidase II cleavage site, indicating that the 40-kDa protein was a lipoprotein. Based on significant homology (31 to 52% identity) of the 40-kDa protein to glycerophosphodiester phosphodiesterases of Escherichia coli (GlpQ), Bacillus subtilis (GlpQ), and Haemophilus influenzae (Hpd; protein D), we have designated this B. hermsii 40-kDa lipoprotein a glycerophosphodiester phosphodiesterase (Gpd) homolog, the first B. hermsii lipoprotein to have a putative functional assignment. A nonlipidated form of the Gpd homolog was overproduced as a fusion protein in E. coli BL21(DE3)(pLysE) and was used to immunize rabbits to generate specific antiserum. Immunoblot analysis with anti-Gpd serum recognized recombinant H. influenzae protein D, and conversely, antiserum to H. influenzae protein D recognized recombinant B. hermsii Gpd (rGpd), indicating antigenic conservation between these proteins. Antiserum to rGpd also identified native Gpd as a constituent of purified outer membrane vesicles prepared from B. hermsii. Screening of other pathogenic spirochetes with anti-rGpd serum revealed the presence of antigenically related proteins in Borrelia burgdorferi, Treponema pallidum, and Leptospira kirschneri. Further sequence analysis both upstream and downstream of the Gpd homolog showed additional homologs of glycerol metabolism, including a glycerol-3-phosphate transporter (GlpT), a glycerol-3-phosphate dehydrogenase (GlpD), and a thioredoxin reductase (TrxB).

Amino Acid Sequence↗
PubMed1991

Identification of a GTP-binding protein in the contact sites between inner and outer mitochondrial membranes.

Whilst investigating whether GTP hydrolysis may be required for the import of preproteins into mitochondria we have found that a GTP-binding protein is located at the contact sites between mitochondrial inner and outer membranes. When mitochondrial outer membranes purified from rat liver were UV-irradiated in the presence of [alpha-32P]GTP, a 52 kDa protein was radiolabelled, whereas [alpha-32P]ATP did not label this protein. GTP-binding proteins were also labelled in the cytosolic and microsomal fractions, but the 52 kDa protein was concentrated in mitochondrial membranes and was the only protein specifically labelled by GTP in these membranes. Fractionation of mitochondrial membrane vesicles into outer membranes, inner membranes and contact sites between outer and inner membranes showed that the GTP-binding activity was highly enriched in contact sites, the location at which preprotein import is believed to occur. A protein of almost identical size was also found to be labelled in mitochondria from yeast.

Adenosine Triphosphate↗
PubMed2001

Analysis of the expression of the putatively virulence-associated neisserial protein RmpM (class 4) in commensal Neisseria and Moraxella catarrhalis strains.

The RmpM protein has been reported to be present only in pathogenic Neisseria species. In the present study we demonstrate that this protein is also present at least in N. lactamica and N. sicca strains. The N. lactamica protein reacts with a RmpM-specific monoclonal antibody (185,H-8), having a molecular mass ( approximately 31 kDa) slightly lower than that of the meningococcal RmpM, and mouse antibodies from sera against outer membrane vesicles from both N. lactamica and N. sicca strains cross-react with the meningococcal RmpM. PCR and hybridization experiments with a complete rmpM probe agree with the immunodetection experiments. Our results strongly suggest that the meningococcal RmpM should not be considered a virulence marker, and the presence of this protein in the commensal species agrees with its role as a structural protein, proposed for the RmpM, which should be considerably conserved in the Neisseria species.

Antibodies, Monoclonal↗
PubMed1983

Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins.

The precursor of cytochrome b(2) (a cytoplasmically-synthesized mitochondrial protein) binds to isolated mitochondria or to isolated outer membrane vesicles. Binding does not require an energized inner membrane, is diminished by trypsin treatment of the membranes and is not observed with the partially processed (intermediate) form of the cytochrome b(2) precursor or with non-mitochondrial proteins. Upon energization of the mitochondria, the bound precursor is imported and cleaved to the mature form. Similar results were obtained with the precursor of citrate synthase. This receptor-like binding activity was present in isolated outer, but not inner membrane. It was solubilized from outer membrane with non-ionic detergent and reconstituted into liposomes.

Journal Article↗
PubMed2003

Differences in immunogenicity and protection in mice and guinea pigs following intranasal immunization with Helicobacter pylori outer membrane antigens.

Mice and guinea pigs were intranasally immunized with either recombinant lipoprotein 20 or Helicobacter pylori outer membrane vesicles (OMV). Cholera toxin was used as mucosal adjuvant. In mice, both vaccines elicited systemic and local IgG responses, which correlated with significantly lower levels of H. pylori colonization. In contrast, only OMV proved immunogenic in guinea pigs, with the development of both systemic and local immune responses. These antibodies did not, however, correlate with protection in these animals, which suggests that vaccine formulation is as important as choice of antigen in the development of an H. pylori vaccine.

Adjuvants, Immunologic↗
PubMed1995

Topological analysis of the Escherichia coli ferrichrome-iron receptor by using monoclonal antibodies.

Ferrichrome-iron transport in Escherichia coli is initiated by the outer membrane receptor FhuA. Thirty-five anti-FhuA monoclonal antibodies (MAbs) were isolated to examine the surface accessibility of FhuA sequences and their contribution to ligand binding. The determinants of 32 of the MAbs were mapped to eight distinct regions in the primary sequence of FhuA by immunoblotting against (i) five internal deletion FhuA proteins and (ii) four FhuA peptides generated by cyanogen bromide cleavage. Two groups of MAbs bound to FhuA in outer membrane vesicles but not to intact cells, indicating that their determinants, located between residues 1 and 20 and 21 and 59, are exposed to the periplasm. One of the 28 strongly immunoblot-reactive MAbs bound to FhuA on intact cells in flow cytometry, indicating that its determinant, located between amino acids 321 and 381, is cell surface exposed. This MAb and four others which in flow cytometry bound to cells expressing FhuA were tested for the ability to block ligand binding. While no MAb inhibited growth promotion by ferrichrome or cell killing by microcin 25, some prevented killing by colicin M and were partially able to inhibit the inactivation of T5 phage. These data provide evidence for spatially distinct ligand binding sites on FhuA. The lack of surface reactivity of most of the immunoblot-reactive MAbs suggests that the majority of FhuA sequences which lie external to the outer membrane may adopt a tightly ordered organization with little accessible linear sequence.

Amino Acid Sequence↗
PubMed1991

A monoclonal antibody against a Pasteurella multocida outer membrane protein protects rabbits and mice against pasteurellosis.

Monoclonal antibodies (MAbs) directed against the 37.5-kDa outer membrane protein were produced by fusing myeloma cells with spleen cells obtained from mice immunized with a pathogenic strain of Pasteurella multocida isolated from a rabbit. Desirable MAbs were selected by enzyme-linked immunosorbent assay, whole-cell radioimmunoprecipitation (WC-RIP), and Western blot (immunoblot) analysis. WC-RIP and Western blot analyses, using MAb 1608 adsorbed with intact P. multocida cells and the eluted MAb, demonstrated that the antigen recognized by this MAb is exposed on the cell surface, is antibody accessible, and has an estimated molecular mass of 37.5 kDa. Treatment of outer membrane vesicles of P. multocida with proteinase K totally abrogated the MAb 1608 activity, indicating that this MAb binds to a protein antigenic determinant. Furthermore, MAb 1608 was nonreactive to purified lipopolysaccharide in Western blot analysis. Passive transfer studies showed that nine rabbits inoculated intranasally with MAb 1608 and homologously challenged intranasally had significantly reduced mortality, severity of pneumonia, prevalence of P. multocida colonization in nonrespiratory organs, and numbers of P. multocida in nasal cavities compared with the controls. Furthermore, the number of P. multocida in lungs was reduced 84,750-fold. Similarly, passive transfer experiments indicated that MAb 1608 protected mice against homologous and heterologous challenges with P. multocida strains bearing the antigenic determinant recognized by MAb 1608. However, no protection was afforded by MAb 1608 when mice were challenged with a P. multocida strain lacking the antigenic determinant recognized by MAb 1608. This study establishes that the 37.5-kDa outer membrane protein is the target for a protective MAb.

Animals↗
PubMed2000

Genetic and immunologic characterization of a novel serotype 4, 15 strain of Neisseria meningitidis.

The porin proteins of Neisseria meningitidis are important components of outer membrane protein (OMP) vaccines. The class 3 porin gene, porB, of a novel serogroup B, serotype 4, 15 isolate from Chile (Ch501) was found to be VR1-4, VR2-15, VR3-15 and VR4-15 by porB variable region (VR) typing. Rabbit immunization studies using outer membrane vesicles revealed immunodominance of individual PorB (class 3) VR epitopes. The predominant anti-Ch501 PorB response was directed to the VR1 epitope. Anti-PorB VR1 mediated killing was suggested by the bactericidal activity of Ch501 anti-sera against a type 4 strain not expressing PorA or class 5 OMPs. Studies that examine the molecular epidemiology of individual porB VRs, and the immune responses to PorB epitopes, may contribute to the development of broadly protective group B meningococcal vaccines.

Animals↗