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PubMed1990

Comparison of methods for the analysis of outer membrane antigens of Neisseria meningitidis by western blotting.

The method of extraction of outer membrane proteins (OMPs), the conditions of electrophoretic transfer, and the conditions of antibody binding, were compared in Western blotting studies of Neisseria meningitidis outer membrane antigens. The OMP profiles obtained by SDS-PAGE of outer membrane vesicles extracted with lithium chloride/sodium acetate were compared with profiles obtained by Sarkosyl extraction; these profiles were further compared with the patterns obtained by 125I-labelling of surface-exposed proteins. Sarkosyl extracts gave profiles most closely resembling those of 125I-labelled whole-cells and gave the best resolution of the major proteins. After transfer in Tris-glycine-methanol buffer some proteins, including the major proteins, were not completely transferred and remained in the gel, with the class 2/3 and 5 proteins not effectively detected on nitrocellulose by amido black staining. There was weak antibody recognition of the class 1 and 4 proteins but good recognition of lipooligosaccharide (LOS) and H8 antigen. Empigen BB had no effect on renaturation of the class 1 protein. When 0.1% SDS was incorporated in the same buffer all of the proteins were removed from the gel, and although the major proteins bound to nitrocellulose other proteins did not. There was weak antibody recognition of the class 1 and 4 proteins, stronger reaction to the class 5 protein, but no recognition of the class 2 protein, LOS or H8 antigen, Empigen BB slightly enhanced antibody recognition of the class 1 protein. After transfer in Tris-glycine buffer, all the major proteins were transferred and bound to nitrocellulose and, other than the class 2 protein, were recognised by antibody, both in the presence or absence of Empigen BB, as were LOS and the H8 antigen. Differences existed in the patterns of antibody recognition between the lithium and the Sarkosyl extracts; additional proteins were recognised in the lithium extracts. The surface-labelling studies indicated, however, that some of these proteins were not surface-exposed. Some minor proteins appeared to be more highly immunogenic than the major proteins.

Acetates↗
PubMed2005

Effects of the Escherichia coli toxin cytolysin A on mucosal immunostimulation via epithelial Ca2+ signalling and Toll-like receptor 4.

Epithelial cells are vital to sense the presence of bacteria, thereby initiating a proper innate immune response. This occurs via different mechanisms, e.g. recognition by pattern recognition receptors (TLR), or alteration of the cellular Ca2+ homeostasis. The Escherichia coli toxin cytolysin A (ClyA) is naturally delivered to target cells as active pore assemblies within outer membrane vesicles (OMVs), and we here investigate a possible role of ClyA-containing OMVs (ClyA+ (OMV)) for induction of proinflammatory responses via the above-mentioned mechanisms. We report that low, sublytic concentrations of ClyA+ (OMV) affect the Ca2+ homeostasis in epithelial cells by induction of slow, intracellular Ca2+ oscillations, while increased concentrations act cytolytically. Thus, ClyA belongs to the novel group of pore-forming toxins shown to elicit such biphasic responses. Ca2+ waves in the minute range have been shown to regulate gene transcription of, e.g. interleukin (IL)-6 and -8. While the periodicity of ClyA+ (OMV)-induced Ca2+ waves (22.9 +/- 0.9 min) fail to induce an IL-8 response, our data fit to the general concept of frequency-specific gene expression. Molecular investigations of the signal transduction pathway reveals that ClyA+ (OMV) utilize a different one as compared with those previously reported for other toxins causing Ca2+ waves. The ClyA protein per se and ClyA pore assemblies are non-immunogenic, while lipopolysaccharide present on the OMVs induces a TLR4-dependent proinflammatory response as expected. Additional membrane components of the OMV, e.g. OmpW, was also found to elicit proinflammatory responses that was independent of TLR4 and Ca2+ signalling.

Animals↗
PubMed1983

The periplasmic maltose-binding protein modifies the channel-forming characteristics of maltoporin.

Maltoporin, a protein spanning Escherichia coli outer membranes, modifies electrical conductance of membranes due to its channel-forming properties. This observation was made by conductance measurements across planar bilayers which were derived from unextracted, isolated outer membrane vesicles using a porin-deficient E. coli strain. Alternatively, proteoliposomes reconstituted with detergent-solubilized homogeneous maltoporin and phospholipids were used. With either membrane preparation, channel conductance was observed, although no discrete conductance levels were detected. The presence of lipopolysaccharide, a bacterial glycolipid, was not required, nor did it affect channel activity. In the presence of the water-soluble periplasmic maltose-binding protein, conductance fluctuations occurred in discrete steps, demonstrating opening and closing events of channels. Multiple step sizes (1/3, 2/3 and 1 ns in 1 M KCl) in single channel traces suggest cooperative opening and closing of up to three channels. The action of maltose-binding protein is highly asymmetrical, and its affinity to maltoporin is very high (KD = 1.5 X 10(-7) M). Association of maltose-binding protein to maltoporin shifts, for a given polarity, the equilibrium between open and closed states in favour of closed states. This result matches earlier in vivo studies, and supports the physiological significance of the observations made.

ATP-Binding Cassette Transporters↗
PubMed1995

Outer membrane-associated deoxyribonuclease activity of Porphyromonas gingivalis.

Extracellular outer membrane vesicles which are produced by Gram-negative bacteria may enclose deoxyribonucleic acid (DNA). While characterizing vesicles of Porphyromonas gingivalis, it was found that they do not contain detectable amount of DNA. It was also shown that the presence of deoxyribonuclease activity on whole cells and vesicles can degrade plasmidic and linear DNA. Deoxyribonuclease activity was also demonstrated in several other Gram-negative oral bacterial species. The nuclease activity of P. gingivalis was further characterized. When deoxyribonuclease activity was analyzed by zymography, only one active band was detected under the conditions tested. This nuclease enzyme showed a molecular weight of approximately 50 kDa. The activity was inhibited by 5 mM ZnCl2 or 100 mM EDTA whereas Mg2+ or Ca2+ ions were not required for activity. Activity was totally destroyed by treatment at 70 degrees C for 15 min. Although the enzyme may participate in virulence or provide nucleic acid precursors for bacterial growth, its exact role is still unknown.

Journal Article↗
PubMed1994

Combination of DTP and Haemophilus influenzae type b conjugate vaccines can affect laboratory evaluation of potency and immunogenicity.

A commonly used Diphtheria-Tetanus-Pertussis (whole-cell) vaccine was combined with each of three different Haemophilus influenzae type b (Hib) capsular polysaccharide vaccines. Each Hib vaccine incorporated one of three different protein conjugates: tetanus toxoid, diphtheria CRM197 toxoid or group B Neisseria meningitidis outer membrane vesicles. The effects of these combinations on the subsequent laboratory control testing were examined. The addition of the Hib vaccines had no significant effect on the reactogenicity or the potency of the whole-cell pertussis component. The potency of, and antibody responses to, the diphtheria component were also unaffected in all three combinations. However, combination with the Hib vaccine comprising polysaccharide conjugated to tetanus toxoid had dramatic effects on tetanus potency and immunogenicity when assayed in mice. This combination resulted in a five-fold potentiation of the tetanus potency and a similarly large increase in the antibody responses to tetanus toxin and toxoid. The level of the antibody response to the Hib polysaccharide in this vaccine was also elevated, more than 20-fold, as a result of the combination. Such phenomena were not evident with combinations involving the other two Hib vaccines. These results have implications for the control testing of combined vaccines containing a whole-cell pertussis component and Hib polysaccharide-tetanus protein conjugate vaccine.

Animals↗
PubMed1996

Blocking of iron uptake from transferrin by antibodies against the transferrin binding proteins in Neisseria meningitidis.

Neisseria meningitidis, when cultured in iron-restricted environments, synthesises new outer-membrane proteins, many of which are necessary for their survival and growth. Some of these proteins e.g. transferrin-binding proteins 1 and 2 (Tbp1 and Tbp2), are required for the acquisition of iron from transferrin and are examples of important iron-regulated meningococcal surface antigens which are not expressed after growth in common laboratory media. The antigenicity and antigenic heterogeneity of these proteins have been extensively studied, and the bactericidal activity of antibodies directed to them have been studied. In this work we analysed the ability of such antibodies to inhibit transferrin binding and to block iron uptake from human transferrin. Antisera from mice immunized with either meningococcal outer membrane vesicles, purified Tbp1/2 complexes, or purified Tbp2, were incorporated in radiolabeled-iron uptake assays. Uptake was blocked by more than 80% in the homologous strains, but blocked much less efficiently in some heterologous strains, correlating well with inhibition of transferrin binding and with an inhibitory effect on bacterial growth. Inhibition of iron uptake from citrate was unaffected which suggests that this effect is due to antibodies against the components of the transferrin binding system, specially Tbp2. Our results support the importance of these proteins and their suitability to be considered in the development of effective vaccines against serogroup B meningococci.

Antibodies, Bacterial↗
PubMed1995

Cytological aspects of resistance to potassium tellurite conferred on Pseudomonas cells by plasmids.

The ultrastructure of strains Pseudomonas putida BS228 and Pseudomonas aeruginosa ML4262 harboring plasmids pBS10, pBS31, and pBS221, which determine resistance to potassium tellurite, was studied. Bacteria were grown in media containing increasing concentrations of potassium tellurite. Crystalline structures containing tellurium appeared in their periplasmic space. The dynamics of crystal growth was studied. Crystals were released into the medium by pinching off of the outer membrane vesicles containing growing crystals. A possible mechanism of this process was described; cytobiochemical peculiarities were discussed.

Crystallization↗
PubMed1989

Hemophilus influenzae adherence to and absorption from the nasal mucosa of guinea pigs.

The adherence to and the absorption from nasal mucosal epithelium of type b and non-typeable strains of Hemophilus influenzae were studied in guinea pigs. H. influenzae bacteria were administered to the nose as 4 x 10(7) organisms in 1.0 ml saline. The outer membrane vesicles (OMV) of H. influenzae were used in the absorption study. Scanning electron microscopic studies showed that H. influenzae cells attached to both ciliated and non-ciliated cells of the nasal mucosa. Histological studies showed that OMV were absorbed through the nasal mucosa, mainly from ciliated cells. Further, penetration of OMV through the nasal mucosa of immunized animals was reduced when compared with that of non-immunized ones.

Animals↗
PubMed2006

Ultrastructural analysis of the extracellular matter secreted by the psychrotolerant bacterium Pseudoalteromonas antarctica NF3.

The psychrotolerant strain Pseudoalteromonas antarctica NF3, a Gram-negative bacterium isolated from muddy soil samples of Antarctica, secretes large amounts of a mucoid exopolymer with a high protein content. It has self-assembly properties and capacity to coat and protect liposomes against surfactants. We examined the ultrastructure of P. antarctica and the extracellular matter it secretes by transmission electron microscopy (TEM) after high-pressure freezing, freeze substitution (HPF-FS), and Epon embedding, and compared this with information obtained by conventional methods. The improvements brought about by HPF-FS to the ultrastructural preservation of the extracellular matter allowed us to establish for the first time, in P. antarctica NF3, the presence of two components: a large amount of cell-derived outer membrane vesicles containing proteins and a capsular polymer around the cells.

Extracellular Space↗
PubMed1993

A constitutive form of heat-shock protein 70 is located in the outer membranes of mitochondria from rat liver.

HSP73, the constitutive form of heat-shock protein 70, has been implicated in the translocation of preproteins across the mitochondrial membranes, being required for maintaining mitochondrial preproteins in an import competent conformation. Here we report that highly purified mitochondrial outer membranes contain a protein indistinguishable from HSP73 as a tightly associated peripheral component of the membrane. This membrane form of HSP73 was photolabelled with [alpha-32P]ATP and could be released from the outer membrane with sodium carbonate, but not after incubation of the membranes with salt or with ATP. A sensitive immunoassay with an anti-HSP73 monoclonal antibody, revealed the association of HSP73 with mitochondrial outer membrane vesicles at a level similar to that of preprotein import receptors.

Adenosine Triphosphate↗
PubMed2005

Virulence factors, pathogenesis and vaccine protection in cholera and ETEC diarrhea.

Recent work has provided new insights into the pathogenesis of the potentially life-threatening diarrheas caused by Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC): a new mechanism (post-translational degradation), which is involved in the control of cholera toxin expression, has been discovered. Recent evidence also suggests that vibrios upregulate cholera toxin expression in response to intestinal fluid components, and enterotoxin-carrying bacterial outer membrane vesicles might have a function in ETEC pathogenesis. An important role of the environment is supported by the correlation between cholera incidence and elevated sea surface temperature, which supports the notion that the zooplankton is a V. cholerae reservoir. Additionally, environmental lytic cholera phages could influence cholera seasonality by 'terminating' the seasonal epidemic. Finally, the strong herd immunity elicited by an oral cholera vaccine indicates that cholera vaccination could have a significant public health impact.

Animals↗
PubMed2004

Immunogenicity of meningococcal PorA formulations encapsulated in biodegradable microspheres.

The purpose of our study was to investigate the possibility to microencapsulate liposomes and meningococcal outer membrane vesicles (OMV), both containing neisserial pore protein A (PorA), in biodegradable dextran- and mannan-based microspheres and to study the immunogenicity of the encapsulated PorA formulations. PorA-liposomes and OMV were encapsulated in dextran- or mannan-based microspheres by using an aqueous two-phase system consisting of a polyethylene glycol solution and a methacrylated dextran or mannan solution. The formulations were characterized for size distribution, PorA structure and antigen recovery after release. Calcein-containing model liposomes were used to establish the encapsulation efficiency and release profiles from both types of microspheres. The immunogenicity of the PorA-containing formulations was determined in mice after subcutaneous immunization. Liposomes were encapsulated in dextran and mannan microspheres with a high efficiency (70-90%). Calcein liposomes, after a 5-day lag period, exhibited apparent zero-order release kinetics from both types of microspheres between Days 5 and 10 of incubation in vitro. The total release was 80 and 100% from mannan and dextran microspheres, respectively. The trimeric PorA conformation was preserved in the released liposomes and OMV and the antigen was partly recovered. The immunogenicity of PorA-liposomes and OMV encapsulated in dextran or mannan microspheres was preserved. In conclusion, PorA-liposomes and OMV could be encapsulated in dextran- and mannan-based microspheres with high efficiency. The immunogenicity of encapsulated antigen was preserved.

Animals↗
PubMed2003

Direct isolation of recombinant human antibodies against group B Neisseria meningitidis from scFv expression libraries.

The successful generation of human antibodies from large nai;ve antibody libraries requires iterative selection steps. Here, we describe a new and fast method for the isolation of high affinity antibodies directly from human single chain Fv antibody (scFv) expression libraries. Escherichia coli scFv expression libraries were made from peripheral blood lymphocytes from four individuals vaccinated with group B Neisseria meningitidis outer membrane vesicle (OMV) vaccine. Forty thousand clones were directly screened for antibodies binding N. meningitidis strain 44/76 (B:15:P1.7,16). Of 430 specific clones detected, 225 candidates were isolated and re-screened against the N. meningitidis strains NZ-98/254 (B:4:P1.7b,4) giving 4% cross-reactive clones. Antibodies were further characterized by DNA sequencing, ELISA and surface plasmon resonance (SPR) analysis, showing broad V-gene diversity and nanomolar scFv affinities. Antibodies derived by this method may assist in the discovery and development of new vaccine antigens as well as therapeutic antibody agents for the treatment of meningococcal diseases.

Amino Acid Sequence↗
PubMed2005

Crystal structure of an Anti-meningococcal subtype P1.4 PorA antibody provides basis for peptide-vaccine design.

In various western countries, subtype P1.4 of Neisseria meningitidis serogroup B causes the greatest incidence of meningococcal disease. To investigate the molecular recognition of this subtype, we crystallised a peptide (P1HVVVNNKVATH(P11)), corresponding to the subtype P1.4 epitope sequence of outer membrane protein PorA, in complex with a Fab fragment of the bactericidal antibody MN20B9.34 directed against this epitope. Structure determination at 1.95 A resolution revealed a unique complex of one P1.4 antigen peptide bound to two identical Fab fragments. One Fab recognises the putative epitope residues in a 2:2 type I beta-turn at residues P5NNKV(P8), whereas the other Fab binds the C-terminal residues of the peptide that we consider a crystallisation artefact. Interestingly, recognition of the P1.4 epitope peptide is mediated almost exclusively through the complementarity-determining regions of the heavy chain. We exploited the observed turn conformation for designing conformationally restricted cyclic peptides for use as a peptide vaccine. The conformational stability of the two peptide designs was assessed by molecular dynamics simulations. Unlike the linear peptide, both cyclic peptides, conjugated to tetanus toxoid as a carrier protein, elicited antibody responses in mice that recognised meningococci of subtype P1.7-2,4. Serum bactericidal assays showed that some, but not all, of the sera induced with the cyclic peptide conjugates could activate the complement system with titres that were very high compared to the titres induced by complete PorA protein in its native conformation administered in outer membrane vesicles.

Animals↗
PubMed2003

Porphyromonas gingivalis induces murine macrophage foam cell formation.

Atherosclerosis is a complex pathologic process initialed by the formation of cholesterol-rich plaque. Macrophages play a central role in the development of atherosclerosis, specifically in the initial accumulation of cholesterol in the arterial wall. It has been suggested that infection and chronic inflammatory conditions such as periodontitis may influence the atherosclerosis process. Porphyromonas gingivalis, one of the major pathogens involved in periodontitis, has been detected in human atheromas, suggesting that P. gingivalis infection may be associated with atherosclerosis. However, a causal relationship between this pathogen and the disease process has not yet been established. The purpose of the present investigation was to determine whether P. gingivalis could induce macrophages to form foam cells using the murine macrophage cell line (J774) as a model system. For inocula smaller than one bacterium per ten cells, P. gingivalis 381, as well as its lipopolysaccharide (LPS), induced foam cell formation of macrophages when cultured in the presence of human low-density lipoprotein (LDL). Infection of macrophages with increasing doses of P. gingivalis resulted in higher levels of foam cell formation. More than 70% of the cultured macrophages form cholesterol ester droplet-rich cells in the presence of 100 mug/ml of LDL when the inocula was more than 10 bacteria per cell. Low concentrations of P. gingivalis outer membrane vesicles also induced foam cell formation in the presence of LDL. In addition, it was demonstrated that P. gingivalis LPS alone was able to induce macrophage foam cell formation. P. gingivalis and its vesicles not only promoted LDL binding to macrophages but also induced macrophages to modify native LDL, which plays an important role in foam cell formation and the pathogenesis of atherosclerosis. Therefore, P. gingivalis cells or its vesicles released from periodontal lesions into the circulation may deliver virulence factor(s) such as LPS to the arterial wall to initiate or promote foam cell formation in macrophages and contribute to atheroma development.

Animals↗
PubMed2003

Role of dentilisin in Treponema denticola epithelial cell layer penetration.

Treponema denticola is an oral anaerobic spirochete implicated in periodontal diseases. The chymotrypsin-like protease, dentilisin (PrtP), has been suggested to be an important virulence factor of T. denticola. In this study, we examined the role of dentilisin in T. denticola epithelial monolayer penetration by comparing the wild type and prtP mutant. Wild-type T. denticola can disrupt transepithelial resistance (TER) and substantially penetrate the HEp-2 cell layer. The prtP mutant altered the monolayer only slightly and penetrated the Hep-2 layer in very low numbers. The membrane fraction of wild-type T. denticola is able to complement the prtP mutant in monolayer penetration, while the comparable fraction from the mutant has no such effect. Immunofluorescence studies suggested that wild-type T. denticola altered the TER by likely degrading the tight junctional proteins such as ZO-1. Cytotoxicity was not a major factor in the disruption of TER. The outer membrane vesicles (OMVs) of wild-type T. denticola also disrupted epithelial barrier function and penetrated the epithelial layers. Taken together, these results suggest that T. denticola penetrates the epithelial cell monolayers by altering cellular tight junctions.

Bacterial Proteins↗
PubMed2004

Immunisation schedules for non-replicating nasal vaccines can be made simple by allowing time for development of immunological memory.

Mice immunised intranasally with multiple doses of outer membrane vesicles (OMVs) from group B meningococci developed antibody responses that depended on the interval between doses. High levels of antibodies in saliva and extracts of faeces were induced within 4 weeks after an OMV vaccine had been given at weekly intervals, whereas the antibody responses in these samples were negligible when given four times at 1-day or 1-h intervals, or as one large dose. Only modest responses were obtained in serum after 4 weeks, however, whether the vaccine had been given repeatedly at any schedule, including the 1-week interval, or as one dose. On the other hand, two large doses given 8 weeks apart induced booster antibody responses in both serum and secretions that matched the responses from a second series of the four smaller doses. Intranasal immunisations may thus stimulate immunological memory more rapidly in secretions than in serum. In order to secure adequate systemic responses by a minimum of doses, nasal vaccines should therefore be given at intervals longer than 4 weeks, in harmony with the intervals recommended for injectable vaccines.

Administration, Intranasal↗
PubMed2005

Meningococcal surrogates of protection--serum bactericidal antibody activity.

Despite the availability of anti-microbial agents effective against Neisseria meningitidis, meningococcal disease continues to be a major global health problem, particularly in the very young. Serogroup A meningococci cause large epidemics in sub-Saharan Africa, whilst serogroups B and C organisms are responsible for sporadic cases and localised outbreaks of disease world-wide. For measuring functional activity, the serum bactericidal antibody (SBA) assay is the most important method. It is mediated by antibody and complement resulting in lysis of the bacterial cells. To date the SBA has proved to be the best surrogate of protection for all serogroups. For serogroup C, an SBA titre of either >/ or =4 or > or =8 has being utilised for putatively indicating protection when using either human or baby rabbit complement, respectively. For serogroup B, the proportions of vaccines with > or =4-fold rises in SBA pre- to post-vaccination or SBA titres > or =4 have been correlated with clinical efficacy in trials of outer membrane vesicle (OMV) vaccines in Cuba, Brazil and Norway. SBA activity as a correlate of protection for evaluating the immune response to meningococcal vaccines is described in this review.

Antibodies, Bacterial↗