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PubMed1981

Investigation of the determinants of the survival of Neisseria gonorrhoeae within human polymorphonuclear phagocytes.

In studies aimed at identifying the determinants responsible for the ability of gonococci to survive and grow within human phagocytes, the reduction of intracellular survival of phagocyte-resistant gonococci by prior treatment of the bacteria with homologous antiserum provided two indirect means of testing for possible determinants. First, surface washes of the resistant gonococci and fractions of these extracts were examined for ability to neutralize the above effect of antisera. Second, antisera raised to purified surface components of the resistant organisms were examined for ability to promote intracellular killing. A combination of these methods indicate that the aggressins were not pili but components of the outer membrane. A surface wash of a phagocyte-resistant, pilated strain, BS4 (agar), neutralized the activity of homologous antisera before and after centrifuging at 20 000 g, but pili separated from the supernatant did not. A corresponding supernatant from the surface wash of a phagocyte susceptible strain, BSSH, had little neutralizing ability. Antisera to pili failed to reduce intracellular survival of resistant gonococci, whereas antisera against outer membrane vesicles did so. Finally, the neutralizing activity of the surface wash supernatant was removed by centrifugation after treatment with wheat germ agglutinin, which precipitates outer membrane vesicles.

Antigens, Bacterial↗
PubMed2004

Release of Shiga toxin by membrane vesicles in Shigella dysenteriae serotype 1 strains and in vitro effects of antimicrobials on toxin production and release.

Effects of various antimicrobials on in vitro Shiga toxin production and release by Shigella dysenteriae serotype 1 was investigated in this study with particular reference to the role of outer membrane vesicles in toxin release by the organism. Five antimicrobials, namely nalidixic acid, ciprofloxacin, norfloxacin, fosfomycin and mitomycin C, were chosen for the study and the toxin titre was measured by the reverse passive latex agglutination (RPLA) method using an available kit. Only mitomycin C was found to induce production of Shiga toxin in the bacteria and its release by outer membrane vesicles. The highest titre of toxin was obtained in vesicle fraction suggesting that the vesicles play an important role in the release of Shiga toxin from periplasmic space by the organism.

Anti-Infective Agents↗
PubMed1990

IgG subclass antibodies to serogroup B meningococcal outer membrane antigens following infection and vaccination.

IgG and IgG subclass antibodies to the outer membrane antigens from Neisseria meningitidis (serogroup B, serotype 15:P1.16) were quantitated by an enzyme-linked immunosorbent assay (ELISA) in sera from 40 patients with group B:15:P1.16 meningococcal disease and 24 volunteers immunized with a serotype 15:P1.16 outer membrane vesicle vaccine. A second injection was given 6 weeks after the first immunization. Patient sera obtained two and six weeks after onset of the disease had significantly higher levels of total IgG, IgG1, IgG2, and IgG3 antibodies to the outer membrane antigens than acute sera, convalescent sera from patients with systemic non-meningococcal bacterial infections and sera from healthy controls. The levels of total IgG and IgG1 remained high one and three years later. Sera from the vaccinees showed high levels of total IgG and IgG1 6, 12 and 26 weeks after the first immunization and high levels of IgG3 6 weeks after the second immunization. No increase of IgG2 or IgG4 levels was observed in the postimmunization sera. Immunoblotting of three convalescent sera demonstrated individual patterns of IgG subclass binding to various outer membrane antigens with most distinct binding of IgG1 and IgG3 antibodies to the class I protein, the H.8 lipoprotein and the lipopolysaccharide. Since IgG1 and IgG3 are the most effective antibodies for complement activation and phagocytosis, group B meningococcal disease and immunization with the serotype 15:P1.16 outer membrane vesicle vaccine stimulate production of those IgG subclasses which have the strongest opsonic and bactericidal activity.

Adolescent↗
PubMed1993

Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria.

Nuclear-encoded proteins destined for mitochondria must cross the outer or both outer and inner membranes to reach their final sub-mitochondrial locations. While the inner membrane can translocate preproteins by itself, it is not known whether the outer membrane also contains an endogenous protein translocation activity which can function independently of the inner membrane. To selectively study the protein transport into and across the outer membrane of Neurospora crassa mitochondria, outer membrane vesicles were isolated which were sealed, in a right-side-out orientation, and virtually free of inner membranes. The vesicles were functional in the insertion and assembly of various outer membrane proteins such as porin, MOM19, and MOM22. Like with intact mitochondria, import into isolated outer membranes was dependent on protease-sensitive surface receptors and led to correct folding and membrane integration. The vesicles were also capable of importing a peripheral component of the inner membrane, cytochrome c heme lyase (CCHL), in a receptor-dependent fashion. Thus, the protein translocation machinery of the outer mitochondrial membrane can function as an independent entity which recognizes, inserts, and translocates mitochondrial preproteins of the outer membrane and the intermembrane space. In contrast, proteins which have to be translocated into or across the inner membrane were only specifically bound to the vesicles, but not imported. This suggests that transport of such proteins involves the participation of components of the intermembrane space and/or the inner membrane, and that in these cases the outer membrane translocation machinery has to act in concert with that of the inner membrane.

Biological Transport↗
PubMed2004

The strategy to control New Zealand's epidemic of group B meningococcal disease.

BACKGROUND: In New Zealand today, babies of Pacific ethnicity born in South Auckland have a 1-in-48 chance of contracting meningococcal disease by the time they are 5 years of age. METHODS: The New Zealand government, Chiron Vaccines and the University of Auckland have collaborated to develop and investigate a group B meningococcal vaccine to allow a mass-immunization program to control a prolonged and intense epidemic. Within 3 years, a strain-specific meningococcal outer membrane vesicle vaccine has been developed, and overlapping clinical trials have been undertaken; a report was submitted for regulatory approval within 2 years. An important aspect of the project's strategy was to apply, with physicochemical data, the results of the New Zealand outer membrane vesicle vaccine trials to the parent vaccine produced and evaluated by the Norwegian National Institute of Public Health. Immunogenicity results for the New Zealand vaccine are promising, with the vaccine showing a reactogenicity profile similar to that of the parent vaccine. CONCLUSIONS: Controlling the epidemic depends on delivering an effective vaccine to the individuals at greatest risk, ie, mainly Maori and Pacific populations that previous health programs have struggled to reach. Participation of and partnership with these communities in public health decision-making and vaccine delivery will be critical to a successful immunization program.

Child, Preschool↗
PubMed2003

Meningococcal serogroup B infections: a search for a broadly protective vaccine.

Meningococcal disease is mainly caused by serogroup B in many West European countries. Recently, a highly efficacious vaccine against infections caused by serogroup C has been introduced in the UK and The Netherlands. However, an effective vaccine against serogroup B has not yet become available. Outer membrane vesicle vaccines against serogroup B were previously tested in large Phase III trials but showed a low efficacy in young children. In addition, the high variability of the vaccines' main component, porin A, potentially diminishes its efficacy. Therefore, several approaches in either optimizing these outer membrane vesicle vaccines or searching for novel, highly conserved antigens are currently under investigation. The sequencing of the meningococcal genome has provided new opportunities to detect additional immunogenic epitopes. In this review, the developments in the search for a broadly protective meningococcal serogroup B vaccine will be discussed.

Animals↗
PubMed1989

Characterization of sodium dodecyl sulfate-stable Bacteroides gingivalis proteases by polyacrylamide gel electrophoresis.

Profiles of the proteolytic activities found in Bacteroides gingivalis culture supernatants, outer membranes, vesicles, and cell extracts were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing covalently bound bovine serum albumin. A total of eight distinct bands of proteolytic activity could be detected. Four of these were found in the culture supernatant (P1, P2, P3, and P4). The outer membranes, vesicles, and the cell extract each contained seven major proteolytic bands (P1, P3, P4, P5, P6, P7, and P8). No activity was found in the membrane-free extract, suggesting that the proteases were associated with the cell envelope. With the exception of P7 and P8, all the proteolytic bands were dependent on reducing agents for activity. The eight proteolytic bands were distributed in an identical manner in all four strains of B. gingivalis studied. The effects of protease inhibitors, pH, and heat were determined. Sulfhydryl group reagents and N-alpha-p-tosyl-L-lysine chloromethyl ketone reduced proteolytic activity. The optimum pH was found to be between 7 and 8. A 30-min preincubation at 50 degrees C inactivated the P6, P7, and P8 proteolytic bands. All proteolytic activity was lost after the samples were heated at 75 degrees C for 30 min.

Bacteroides↗
PubMed1989

Application of fourth derivative absorption spectroscopy to protein quantitation during purification.

A method for protein quantitation in the presence of nonprotein cellular components is described. The method is based on measurement of two tryptophan-specific signals in the fourth derivative of the protein's ultraviolet absorption spectrum, a peak at 283 nm and a trough at 288 nm. The amplitude between these two extremes is shown to vary linearly with protein concentration for bovine serum albumin and the outer membrane vesicles of Neissera meningitidis even when these protein solutions are supplemented with enough nucleic acid to completely obscure the parent absorption spectrum of the protein. The utility of this method as an in-process assay during isolation of a protein is demonstrated by comparing estimates of protein content from fourth derivative spectroscopy with those from the Lowry assay for samples at several steps along the isolation pathway for outer membrane vesicles of N. meningitidis. The advantages and limitations of the present method are discussed.

Bacterial Outer Membrane Proteins↗
PubMed1981

Participation of pili and cell wall adhesion in the yeast agglutination activity of Escherichia coli.

Escherichia coli strain 2699 (O6:K13) which had been isolated from a case of urinary tract infection exhibited pili during the stationary phase (24 to 40 h), but not during the exponential phase (4 h), when grown in static broth culture. The bacteria were also piliated when grown for 24 h on agar. They agglutinated Saccharomyces cerevisiae (baker's yeast) in the piliated as well as in the nonpiliated state. The agglutinations were mannose sensitive, i.e., they could be inhibited with 50 mM methyl-alpha-mannoside. The bacteria were first depiliated by shearing and then used for the isolation of outer membrane vesicles with an Omnimixer. Purified pili and outer membranes were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy. The pili could be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis only after treatment at low pH or in saturated guanidine hydrochloride which is typical of the common type 1 pili. Depiliated bacteria, purified pili, and purified outer membranes gave mannose-sensitive agglutination of S. cerevisiae. The findings are discussed with respect to possible mechanisms of cell agglutination.

Agglutinins↗
PubMed1984

Measurement of the rotational behaviour of virus particles during adsorption to the host cell surface.

The rotational diffusion of bacteriophage epsilon 15 was measured before and after virus adsorption to outer membrane vesicles of the host Salmonella anatum. The virus capsid was labeled with eosin isothiocyanate, and the decay of transient dichroism following dye excitation by pulses of plane-polarized light was measured. From the data, the rotational diffusion constant of the unadsorbed virion and its hydrodynamic diameter were estimated and found to be consistent with electron microscopic measurements of the capsid dimensions. Addition of outer membrane vesicles of S. anatum to the virus suspension revealed the immobilization of the virus particles on the membrane surface.

Cell Membrane↗
PubMed1990

Selective extraction of outer-membrane proteins from membrane complexes of Pseudomonas maltophila by chloroform-methanol.

An organic phase partitioning method is described for the selective purification of outer-membrane proteins (OMPs) from the total membrane complex of the opportunistic human and sheep pathogen, Pseudomonas maltophila. SDS-PAGE analysis confirmed that OMPs purified by chloroform-methanol treatment of the total membrane complex were not only identical to OMPs extracted from outer-membrane vesicles separated by sucrose gradient density centrifugation, but also possessed little or non-detectable levels of inner-membrane contaminants. Further analysis by enzyme linked immunosorbent assay (ELISA) and immunoblotting established that OMPs extracted by organic phase partitioning with chloroform-methanol retained antigenicity and serological activity indistinguishable from OMPs that were present in outer-membrane vesicles resolved by isopycnic sucrose density centrifugation of sarkosyl-treated membrane complexes.

Animals↗
PubMed1999

Functional activities and epitope specificity of human and murine antibodies against the class 4 outer membrane protein (Rmp) of Neisseria meningitidis.

Antibodies against the class 4 outer membrane protein (OMP) from Neisseria meningitidis have been purified from sera from vaccinees immunized with the Norwegian meningococcal group B outer membrane vesicle vaccine. The human sera and purified antibodies reacted strongly with the class 4 OMP in immunoblots, whereas experiments with whole bacteria showed only weak reactions, indicating that the antibodies mainly reacted with parts of the class 4 molecule that were not exposed. The purified human anti-class 4 OMP antibodies and the monoclonal antibodies (MAbs) were neither bactericidal nor opsonic against live meningococci. Three new MAbs against the class 4 OMP were generated and compared with other, previously described MAbs. Three linear epitopes in different regions of the class 4 OMP were identified by the reaction of MAbs with synthetic peptides. The MAbs showed no blocking effect on bactericidal activity of MAbs against other OMPs. However, one of the eight purified human anti-class 4 OMP antibody preparations, selected from immunoblot reactions among sera from 27 vaccinees, inhibited at high concentrations the bactericidal effect of a MAb against the class 1 OMP. However, these antibodies were not vaccine induced, as they were present also before vaccination. Therefore, this study gave no evidence that vaccination with a meningococcal outer membrane vesicle vaccine containing the class 4 OMP induces blocking antibodies. Our data indicated that the structure of class 4 OMP does not correspond to standard beta-barrel structures of integral OMPs and that no substantial portion of the OmpA-like C-terminal region of this protein is located at the surface of the outer membrane.

Adult↗
PubMed1993

Cross-reacting serum opsonins to meningococci after vaccination.

The opsonic activity of sera from healthy volunteers immunized with an outer membrane vesicle vaccine prepared from a Neisseria meningitidis (class 3, 44/76, B:15:P1.7,16), characteristic of the present Norwegian epidemic, has been examined. A marked increase in the phagocytosis of class 3 and nontypeable strains of different serogroups, serotypes, and serosubtypes was demonstrated in the presence of postvaccination sera. Sera from vaccinees also caused a significant increase in leukocyte oxidative metabolism as measured by luminol-enhanced chemoluminescence during phagocytosis of class 3 and nontypeable meningococci. An increase in serum opsonins cross-reacting with class 2 (type 2a) meningococci of different serogroups was not observed, suggesting that future meningococcal outer membrane vesicle vaccine preparations should contain both class 2 and class 3 porins in geographic areas where both class 2 and class 3 strains cause disease.

Antibodies, Bacterial↗
PubMed2004

Characterization of a cytotoxic pilin subunit of Xenorhabdus nematophila.

Xenorhabdus nematophila is an insect pathogenic bacterium, known to produce protein toxins that kill the larval host. We have described a cytotoxic pilin subunit of X. nematophila, which is expressed on the cell surface and also secreted in the extracellular medium associated with outer membrane vesicles. A 17kDa pilin subunit was isolated and purified from X. nematophila cell surface. The protein showed cytotoxicity to larval hemocytes of Helicoverpa armigera in an in vitro assay, causing agglutination of the cells, and releasing cytoplasmic enzyme lactate dehydrogenase in the medium. The pilin protein was able to bind to the surface of larval hemocytes. The binding and cytotoxicity of the purified 17kDa protein to hemocytes was inhibited by antiserum raised against the pilin protein. The study demonstrates for the first time a cytotoxic structural subunit of pilin from an entomopathogenic bacterium X. nematophila that is excreted in the extracellular medium with outer membrane vesicles.

Agglutination Tests↗
PubMed2005

Porin A-specific antibody avidity in patients who are convalescing from meningococcal B disease.

Porin A (PorA), which determines the serosubtype of Neisseria meningitidis, is the main antigen of a candidate vaccine against serogroup B meningococci, which has been shown to induce high-avidity antibodies in children. We characterized the immune response of children after convalescing from meningococcal infection with a serosubtype P1.7-2,4 strain. Acute- and convalescent-phase sera of 21 children with meningococcal septic shock caused by strains with PorA subtype P1.7-2,4 were collected. The serum bactericidal antibody titers, IgG isotype distribution, and antibody avidity were measured. We determined whether the differences in avidity of anti-outer membrane vesicle antibodies were PorA specific. Serum bactericidal activity against H44/76 P1.7-2,4 was <4 in all convalescent sera. The IgG isotype distribution of the convalescent sera was dominated by IgG(1), followed by IgG(3), whereas no IgG(2) or IgG(4) was found. The geometric mean avidity index (GMAI) of convalescent sera measured against a strain with the identical subtype as the infective isolate was significantly higher than that against a strain with a heterologous PorA subtype or a PorA-negative mutant strain (57 versus 35 and 23%, respectively; p = 0.005 and p < 0.001). Geometric mean avidity titers were highest for P1.7-2,4, corresponding with the highest GMAI. The GMAI after invasive meningococcal disease was lower than after vaccination of healthy toddlers with a monovalent P1.7-2,4 outer membrane vesicle vaccine.

Antibody Affinity↗
PubMed2000

IgG antibody levels to meningococcal porins in patient sera: comparison of immunoblotting and ELISA measurements.

IgG antibody levels to the meningococcal PorA and PorB proteins in 56 acute and convalescent phase sera from 25 patients with meningococcal disease were compared by immunoblotting and ELISA. Heat-treated outer membrane vesicles from strain 44/76 (B:15:P1.7, 16) served as antigens for immunoblotting, whereas purified P1.7,16 PorA and P15 PorB from the same strain were used as antigens in the ELISA. In the blotting method, IgG binding to the porins was determined by digital scanning of the immunoreactive bands and calculated relative to the PorA binding of a reference serum on each blot. The coefficient of variation for the reference serum was 21.6% (a total of 144 strips) with smaller variations for each day's experiments. Blotting of all 56 sera at the standard 1/200 dilution measured anti-PorA and anti-PorB levels that correlated with those obtained by ELISA (Spearman rank-order correlation coefficient r(s)=0.48; P<0.001). At this dilution, the anti-PorA (r(s)=0.52; P<0. 004) and anti-PorB (r(s)=0.60; P<0.001) levels of the convalescent phase sera (n=29) corresponded with the ELISA measurements, whereas no correlation was found with the results for the acute phase sera, which mostly had low ELISA antibody levels (<2 microg/ml IgG). A corresponding blot analysis of convalescent sera from the seven patients, who had received the 44/76 outer membrane vesicle vaccine, demonstrated a high correlation coefficient for the anti-PorA levels (r(s)=0.95; P<0.001) vs. the ELISA results. No such correlation was observed for the PorB response in these sera, being nine-fold higher than the PorA response, because of a prozone effect on the blots at the standard dilution. However, blotting at a higher serum dilution (1/2000) resulted in anti-PorB levels that also correlated strongly (r(s)=0.93 P<0.001) with the ELISA measurements.

Antigens, Bacterial↗
PubMed1987

Interaction of Escherichia coli and macrophages: alteration by treatment of bacteria with beta-lactam antibiotics.

Antibiotics are known to exert an influence on the host-parasite relationship either by impairment of immunocompetent cells or by alteration of the bacterium, such as changes of surface properties or the production of toxins. The main problem in investigating the effect of antibiotics on the surface properties of bacteria consists in morphological changes of bacteria (round cell or filament formation) after treatment e.g. with beta-lactam antibiotics. These changes of morphology lead to problems in the comparison of such bacterial forms with untreated organisms. Therefore, in this study outer membrane vesicles from bacteria were used as a model to investigate the effect of antibiotics on the surface properties of Escherichia coli with regard to the interaction with mouse peritoneal macrophages tested by chemiluminescence reaction. It could be shown that these membrane vesicles induce a luminol dependent chemiluminescence response. Treatment of E. coli with different beta-lactams lead to an increase of the stimulating properties. The relative effectiveness of certain antibiotics depended on the particular E. coli strain. Analysis of the different adhesions involved in the stimulation of macrophages revealed that only mannose-sensitive adhesins were increased after treatment with beta-lactam antibiotics. No stimulation of the membrane-bound NAD(P)H-oxidase could be found following the reaction with outer membrane vesicles. Even the treatment of bacteria with antibiotics did not evoke such a reaction.

Animals↗