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PubMed1999

flaA mRNA transcription level correlates with Helicobacter pylori colonisation efficiency in gnotobiotic piglets.

In-vivo passage of the gastric pathogen Helicobacter pylori in gnotobiotic piglets results in a greater colonisation efficiency in subsequent infections. The rate of colonisation steadily increases with the number of passages. To determine if this increased efficiency is related to the level of expression of flaA, a gene which encodes the major subunit of flagella, this study evaluated the level of flaA expression at five points during serial in-vivo passage of strain 26695. Semi-quantitative reverse transcriptase polymerase chain reaction and Northern dot-blot analysis of flaA mRNA levels (expressed as a ratio to the level of ureA mRNA) revealed a positive correlation between flaA expression and colonisation efficiency; flaA mRNA levels increased incrementally with in-vivo passage as did colonisation rates. Immunoblots of outer-membrane vesicles from pig-passaged and laboratory-passaged strains also demonstrated marked differences in the amount of flagellin present in poor and efficient colonisers.

Animals↗
PubMed2003

Immune response to native NadA from Neisseria meningitidis and its expression in clinical isolates in Brazil.

A mAb against the NadA protein from Neisseria meningitidis strain 3006 (serosubtype B : 2b : P1.2 : P5.2,8) demonstrated strong bactericidal activity against Brazilian epidemic serogroup B strain N44/89 (B : 4,7 : P1.19,15 : P5.5,7) and a serogroup C strain, IMC 2135 (C : 2a : P1.5,2), but not against another serogroup C strain, N1002/90 (C : 2b : P1.3 : P5.8). The immunogenicity of native NadA in an outer-membrane vesicle (OMV) preparation was also tested. Serum from mice immunized with OMV from serogroup B strain N44/89, which contains the NadA protein, showed bactericidal activity against serogroup B and C strains possessing NadA. In dot-blot analysis of 100 serogroup B and 100 serogroup C isolates from Brazilian patients, the mAb to NadA recognized about 60 % of the samples from both serogroups. The molecular mass of the NadA protein from strain N44/89 determined by mass spectrometry was 37 971 Da and the peptide sequences were identical to those of NadA from N. meningitidis strain MC58.

Amino Acid Sequence↗
PubMed2005

Prevalence of genosubtypes (PorA types) of serogroup B invasive meningococcus in the north of Spain from 2000 to 2003.

The composition of new vaccines against Neisseria meningitidis serogroup B is based on differences in the variable regions VR1 and VR2 of the class 1 outer-membrane protein (PorA) of meningococci. Genosubtyping of 96 N. meningitidis B isolates from blood or cerebrospinal fluid from 2000 to 2003 in the north of Spain allowed characterization of all the strains. Twenty-six genosubtypes or distinct PorA types were obtained. The most prevalent were P1.5-1, 10-8 (20 strains), P1.19, 15 (14 strains), P1.22, 9 (11 strains) and P1.5, 2 (nine strains), while 17 genosubtypes were represented by only one or two strains. The wide diversity of genosubtypes observed and their differences compared with those found in other regions reveals the difficulty in designing a useful outer-membrane vesicle vaccine applicable to different regions of the world.

Bacterial Typing Techniques↗
PubMed1992

Further studies on some physical and biochemical characteristics of asaccharolytic pigmented Bacteroides of feline origin.

The ultrastructure of the appendages of 24 strains of asaccharolytic pigmented Bacteroides spp. of cats was studied by transmission electron microscopy. All strains examined by thin section showed abundant fimbriae, outer membrane vesicles and capsules. Negative staining showed fimbriae which varied from long, fine and wavy in Bact. salivosus and cat Group 2 to shorter, less abundant and thicker fimbriae in cat strains of Bact. gingivalis as well as type strains of Porphyromonas gingivalis and P. asaccharolytica. Capsular material was very thick amorphous in human P. gingivalis, cat strains of Bact. gingivalis and in P. assaccharolytica but fine and fibrillary in preparations of Bact. salivosus and cat Group 2 organisms. Wet india ink preparations showed a large capsule although those of Bact. salivosus and Group 2 appeared largest. Five-day Group 2 broth cultures featured a thick ropy growth consistent with a large accumulation of extracellular slime. Enzymatic activities of the 24 strains measured by API ZYM system as well as the conventional biochemical tests showed it was possible to differentiate reliably Bact. salivosus from the other cat strains of asaccharolytic pigmented Bacteroides species and from human P. gingivalis and P. endodontalis by a combination of these tests. These tests suggest that Bact. salivosus is unlikely to belong to the genus Prevotella. Its place within the genus Porphyromonas is still to be determined.

Animals↗
PubMed2005

Immunoglobulin-A antibodies in upper airway secretions may inhibit intranasal influenza virus replication in mice but not protect against clinical illness.

Mice immunized intranasally with a formalin-inactivated A/PR/8/34 (H1N1) influenza whole virus vaccine adjuvanted with cholera toxin, outer membrane vesicles from group B meningococci or formalin-inactivated whole cell Bordetella pertussis were protected against replication of the homologous virus in the nasal cavity. Only some mice were protected against clinical illness measured as weight loss and lowered body temperature. All mice immunized subcutaneously with one-tenth the intranasal vaccine dose without adjuvant were protected against clinical illness but not against local mucosal viral replication. Replicating virus was primarily found in animals with low concentrations of immunoglobulin (Ig)-A antibodies in saliva regardless of concentrations of IgG antibodies in serum. Clinical illness was seen only in those with low serum antibodies regardless of antibody levels in saliva. Nonreplicating nasal vaccines may not be sufficiently protective unless they also have a substantial influence on systemic immunity.

Adjuvants, Immunologic↗
PubMed1993

Recent approaches to the chemotaxonomy of the Actinobacillus-Haemophilus-Pasteurella group (family Pasteurellaceae).

Many members of the Actinobacillus-Haemophilus-Pasteurella group (family Pasteurellaceae) have been misclassified. This article reviews the chemotaxonomic characters that recently have been provided to improve the taxonomy of Pasteurellaceae. These include fatty acids of whole cells, of lipopolysaccharides and of single colonies, together with sugar contents of whole cells, of whole defatted cells, of lipopolysaccharides and of single colonies. This article also reviews taxonomy aided by distribution of proteins in whole cells and outer membranes, distribution of enzymes in outer membrane vesicles and in whole cells, bacteriolysis induced by ethylenediaminetetraacetic acid and hen eggwhite lysozyme and the distribution of respiratory quinones. Furthermore, an overview of characters obtained through studies on genetic transformation, restriction enzyme analysis, restriction fragment length polymorphism, DNA-DNA hybridization, DNA-rRNA hybridization, and 16S rRNA sequencing is given.

Aggregatibacter actinomycetemcomitans↗
PubMed1991

Analysis of the molecular mass heterogeneity of the transferrin receptor in Neisseria meningitidis and commensal Neisseria.

Peroxidase-conjugated transferrin was used to detect transferrin receptors both in intact outer membrane vesicles (OMVs) from Neisseria species in a dot blot assay, and in SDS-PAGE-separated OMV proteins after transferring to nitrocellulose membranes. All N. meningitidis strains produced transferrin receptors after culturing in either iron sufficiency or iron restriction although expression was higher in the latter case, whereas only six N. lactamica and two N. sicca (among 20 commensal species) were able to bind transferrin. Molecular mass (MM) of the receptors were mainly between 78 kDa and 85 kDa (87.5% of strains), 12.5% had receptors with MM close to 70 kDa, and 5% showed receptors with MM over 85 kDa. Our results confirm the molecular mass heterogeneity of the transferrin receptors in N. meningitidis, completely disagree with the 'universal' 98 kDa receptor proposed by some authors, and show a low expression of the receptor in commensal Neisseria.

Densitometry↗
PubMed1993

Purification of the Neisseria meningitidis transferrin binding protein-2 (TBP2) to homogeneity using column chromatography.

A method for purifying TBP2 from N. meningitidis has been developed using affinity chromatography on Tf-agarose followed by ion exchange chromatography; the Tf-binding activity of fractions was evaluated by a dot-blot technique. This method allowed the purification of the TBP2 in milder conditions than those used previously and to a high degree of homogeneity as was demonstrated by Coomassie brilliant blue or Silver staining after SDS-PAGE electrophoresis. The SDS-PAGE profile of the material obtained in the Tf-agarose affinity chromatography step shows only two detectable proteins, identified as the TBP1 and the TBP2, with a small amount of contamination. Passage through a MonoQ HR anion exchange column, allowed the isolation of TBP2 in the absence of TBP1. Our results demonstrate the conservation of the antigenic reactivity of this protein, which produces monospecific serum with the antibodies elicited reacting exclusively with the TBP2 in whole outer membrane vesicles.

Carrier Proteins↗
PubMed1993

Porphyromonas gingivalis invades oral epithelial cells in vitro.

The aim of the present study was to analyze the adhesive and invasive potential of a number of P. gingivalis strains, in an in vitro system utilizing cultures of human oral epithelial cells (KB cell line, ATCC CCL 17). P. gingivalis strains W50 and FDC 381 (laboratory strains) and OMGS 1738, 1743 and 1439 (clinical isolates) as well as E. coli strain HB 101 (non-adhering, non-invasive control) were used. Adherence was assessed by means of scintillation counting and light microscopy, after incubation of radiolabelled bacteria with epithelial cells. In the invasion assay, monolayers were infected with the P. gingivalis and E. coli strains and further incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). Invasion was evaluated by (i) assessing presence of bacteria surviving the antibiotic treatment, and (ii) electron microscopy. All P. gingivalis strains adhered to and entered into the oral epithelial cells. After 3 hours of incubation, bacteria were frequently identified intracellularly by means of electron microscopy. The cellular membranes, encapsulating the microorganisms in early stages of the invasive process, appeared later to disintegrate. The presence of coated pits on the epithelial cell surfaces suggested that internalization of P. gingivalis was associated with receptor-mediated endocytosis (RME). Formation of outer membrane vesicles (blebs) by intracellular bacteria indicated that internalized P. gingivalis was able to retain its viability. E. coli strain HB 101 neither adhered to nor invaded epithelial cells.

Bacterial Adhesion↗
PubMed1994

Porphyromonas gingivalis invades human pocket epithelium in vitro.

The present study examined the adhesive and invasive potential of Porphyromonas gingivalis interacting with human pocket epithelium in vitro. Pocket epithelial tissue, obtained during periodontal surgery of patients with advanced periodontal disease, generated a stratified epithelium in culture. P. gingivalis strains W50 and FDC 381 (laboratory strains), OMGS 712, 1439, 1738, 1739 and 1743 (clinical isolates) as well as Escherichia coli strain HB101 (non-adhering control) were tested with respect to epithelial adhesion and invasion. Adhesion was quantitated by scintillation spectrometry after incubation of radiolabeled bacteria with epithelial cells. The invasive ability of P. gingivalis was measured by means of an antibiotic protection assay. The epithelial multilayers were infected with the test and control strains and subsequently incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). The number of internalized bacteria surviving the antibiotic treatment was assessed after plating lyzed epithelial cells on culture media. All tested P. gingivalis strains adhered to and entered pocket epithelial cells. However, considerable variation in their adhesive and invasive potential was observed. E. coli strain HB101 did not adhere or invade. Transmission electron microscopy revealed that internalization of P. gingivalis was preceded by formation of microvilli and coated pits on the epithelial cell surfaces. Intracellular bacteria were most frequently surrounded by endosomal membranes; however, bacteria devoid of such membranes were also seen. Release of outer membrane vesicles (blebs) by internalized P. gingivalis was observed. These results support and extend previous work from this laboratory which demonstrated invasion of a human oral epithelial cell-line (KB) by P. gingivalis.

Bacterial Adhesion↗
PubMed2004

Interaction of Porphyromonas gingivalis with low-density lipoproteins: implications for a role for periodontitis in atherosclerosis.

OBJECTIVE: The association of periodontitis with atherosclerosis has been suggested from epidemiological studies. Recently, we have reported that macrophages stimulated by Porphyromonas gingivalis formed foam cells in the presence of low-density lipoproteins (LDL). In this study, we examined the direct interactions between LDL and P. gingivalis. METHODS: We investigated the aggregation of LDL with P. gingivalis and its outer membrane vesicles (OMVs), degradation of the apo B-100 protein of LDL by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses, as well as the effects of protease inhibitors or activators on the mobility of LDL by agarose gel shift assays. The binding of P. gingivalis or its OMVs with LDL was demonstrated by western blot analysis. We also examined whether or not the aggregated LDL induced foam cell formation from murine macrophages. RESULTS: LDL was aggregated in a dose-dependent manner with P. gingivalis and its OMVs. Moreover, degradation of the apo B-100 protein of LDL was directly demonstrated in the presence of P. gingivalis or its OMVs. Furthermore, the gel shift assays indicated that the mobility of LDL was increased in the presence of P. gingivalis. This alteration was attenuated in the presence of the protease inhibitors TLCK and leupeptin and increased in the presence of reducing agents. Moreover, LDL was bound to specific proteins of P. gingivalis suggesting that these proteins may also play a role in aggregation. Finally, the aggregated LDL induced murine macrophages to form foam cells. CONCLUSIONS: These results suggest that P. gingivalis may stimulate foam cell formation, in part, by aggregating LDL by proteolysis of apo B-100.

Adhesins, Bacterial↗
PubMed1983

Binding of metallic ions to the outer membrane of Escherichia coli.

The binding of metal ions by the outer membrane of Escherichia coli K-12 strain AB264 was investigated by using outer membrane obtained after Triton X-100 extraction of purified cell envelopes. Binding studies, conducted under saturating conditions, indicated a selective trapping of certain metallic ions. Low-dose electron microscopy of metal-loaded samples revealed an aggregative deposition of lead on one surface of the membrane which suggests that at least one distinctive binding site is asymmetrically arranged in these outer membrane vesicles.

Binding Sites↗
PubMed1990

Branhamella catarrhalis: an organism gaining respect as a pathogen.

Branhamella catarrhalis was formerly regarded as a common, essentially harmless inhabitant of the pharynx. This misapprehension was caused, in part, by confusion with another pharyngeal resident, Neisseria cinerea. The two organisms can now be differentiated by the positive reactions of B. catarrhalis in tests for nitrate reduction and hydrolysis of tributyrin and DNase. B. catarrhalis is currently recognized as the third most frequent cause of acute otitis media and acute sinusitis in young children. It often causes acute exacerbations of chronic bronchopulmonary disease in older or immunocompromised adults and is incriminated occasionally in meningitis, endocarditis, bacteremia, conjunctivitis, keratitis, and urogenital infections. Virulence-associated factors, such as pili, capsules, outer membrane vesicles, iron acquisition proteins, histamine-synthesizing ability, resistance to the bactericidal action of normal human serum, and binding to the C1q complement component, have been identified in some strains. beta-Lactamase producing strains, first detected in 1976, have risen to approximately 75% worldwide. Thus far, however, practically all American strains of B. catarrhalis remain susceptible to alternative antibiotics. A possible selective advantage of recent isolates is their reportedly heightened tendency for adherence to oropharyngeal cells from patients with chronic bronchopulmonary disease.

Bacterial Infections↗
PubMed1982

Monoclonal antibodies directed against a cell surface-exposed outer membrane protein of Haemophilus influenzae type b.

Monoclonal antibodies directed against several different Haemophilus influenzae type b outer membrane proteins with apparent molecular weights of 45,000, 39,000, and 37,000 were identified by a radioimmunoprecipitation method. Five monoclonal antibodies, including both immunoglobulin G and M isotypes, were specific for the same H. influenzae type b major outer membrane protein (39,000 molecular weight). One of these immunoglobulin G monoclonal antibodies (6A2) was shown to be directed against a cell surface-exposed antigenic determinant of the 39,000-molecular-weight protein, whereas the other monoclonal antibodies directed against this same protein were apparently specific for antigenic determinants not exposed on the H. Influenzae type b cell surface. The cell surface-exposed protein antigenic determinant recognized by monoclonal antibody 6A2 was not unique to the H. influenzae type b strain used as the source of outer membrane vesicles for generating immune spleen cells, but was found in a majority of independently isolated strains of H. influenzae type b. These data indicate that there is antigenic cross-reactivity among H. influenzae type b strains with regard to cell surface-exposed proteins.

Antibodies, Monoclonal↗
PubMed1982

Protection against group B Neisseria meningitidis disease: effect of serogroup B polysaccharide and polymyxin B on immunogenicity of serotype protein preparations.

The inability to prepare an effective polysaccharide vaccine against group B Neisseria meningitidis was the impetus for these studies. Outer membrane protein vaccines used in our initial studies failed to induce bactericidal antibodies in humans. The particulate nature of these vaccines may have led to their clearance before effective immune stimulation. Less denaturing procedures, therefore, were developed for preparation of serotype 2 protein-containing vaccines. These procedures included isolation of naturally released outer membrane vesicles and selective removal of lipopolysaccharide from the vesicles by the nonionic detergent Brij-96. The resultant protein vaccines were evaluated with and without noncovalently complexed group B meningococcal polysaccharide or polymyxin B sulfate or both. The new vaccines were at least 10-fold more immunogenic in mice and guinea pigs than the previous vaccines when assayed for bactericidal and enzyme-linked immunosorbent assay antibodies. The protein vaccines alone protected guinea pigs from intrachamber infection, and a single 0.1-microgram injection prevented meningococcal bacteremia in mice. Addition of group B polysaccharide to the protein significantly improved the immunogenicity of the protein, and this combined vaccine showed a greater protective effect. Polymyxin B generally reduced the immunogenicity of the vaccines in both mice and guinea pigs.

Animals↗
PubMed1985

Actinlike material in Pseudomonas aeruginosa.

Actinlike material was obtained from disrupted Pseudomonas aeruginosa cells by a modification of the method of Hancock and Nikaido (J. Bacteriol. 136:381-390) for isolating outer membrane vesicles. Pelleted membranes were dissolved in Laemmli sample buffer and electrophoresed in parallel lanes with purified rabbit skeletal muscle actin. The bacterial preparation migrated similarly to rabbit skeletal muscle actin. A doublet band was detectable by affinity-purified antiactin antibody in a passive transfer immunoblot. Molecular weight of the actinlike protein doublet was 60,000 to 63,000 as determined by linear regression analysis of Bio-Rad low-molecular-weight standards run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transmission electron microscopy of the actinlike material in high salt concentrations revealed 10- to 14-nm filaments of various lengths. Despite its ability to form filaments and to react with a polyclonal rabbit antiactin antibody, the bacterial filaments did not bind the S-1 fragment of heavy meromyosin.

Actins↗
PubMed1988

Proteins that appear to be associated with pili in Neisseria gonorrhoeae.

Pili of Neisseria gonorrhoeae are thought to be composed entirely of identical subunits, called pilin, that self-assemble in vitro. Previous pilus purification methods have relied on this latter point, and dissociation and reassociation of pilin subunits has yielded pilin preparations of high purity. Such a procedure could result in the loss of any pilus-associated proteins. We have developed a procedure for the isolation of intact native pili in a deoxycholate-urea buffer in which the pili are fractionated on the basis of size and hydrophobicity. Electron microscopy indicates that the pili are largely free from outer membrane vesicles and other cellular material. Electrophoretic analysis has shown that a number of proteins copurify with pilin. Antibodies to these proteins could be removed from an antiserum against whole piliated cells by absorption with piliated cells but not by absorption with nonpiliated cells. Hence, our results indicate that these proteins could be pilus associated.

Bacterial Proteins↗
PubMed1992

Inactivation of human serum bactericidal activity by a trypsinlike protease isolated from Porphyromonas gingivalis.

A protease was isolated from an outer membrane vesicle preparation of Porphyromonas gingivalis ATCC 33277 and assessed for its ability to inactivate the bactericidal activity of normal human serum. The enzyme, which was activated by reducing agents, was found to be a trypsinlike protease with a molecular mass of approximately 80 kDa. Prior to being tested in the bactericidal assay, pooled human serum was preincubated with the partially purified enzyme. Under conditions in which the trypsinlike protease was activated, a strong reduction of the serum bactericidal activity against Capnocytophaga ochracea was noted. On the other hand, no reduction of the bactericidal action of serum was observed when the serum-protease mixture was preincubated in the presence of an inhibitor of the enzyme. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protease was shown to degrade immunoglobulins G and M as well as complement factor C3. This study confirms the previous hypothesis that the proteases of P. gingivalis can interfere with the protective action of human serum.

Blood Bactericidal Activity↗