N-acetyl-D-glucosamine specific hemagglutinin receptor of Vibrio cholerae O1 in chicken erythrocyte membranes.

N-Acetyl-D-glucosamine specific cell-associated hemagglutinin (HA)/lectin, previously purified from a strain of Vibrio cholerae O1, had been established as an adhesin molecule of V. cholerae O1 cells. This communication records the isolation and purification of the glycoprotein receptor of the N-acetyl-D-glucosamine specific HA of the V. cholerae O1 strain from chicken erythrocyte membranes. The most salient feature of this study is that the pretreatment of partially purified glycoprotein with purified HA could completely inhibit the hemagglutinating activity of the V. cholerae O1 strain with chicken erythrocytes.

Adhesion and invasion of a mutant Shigella flexneri to an eukaryotic cell line in absence of the 220-kb virulence plasmid.

A Shigella flexneri strain, cured of the large 220-kb virulence plasmid, expresses adhering and invading ability in confluent monolayers of HeLa cells similar to its parent strain. Invasion by both the parent and the cured strains resulted in alteration of the monomeric actin (G) in the total actin pool of HeLa cells. Other indicators of invasive characteristics of virulent Shigella strains such as production of keratoconjunctivitis in guinea pig eye in vivo, Congo red binding and expression of contact hemolysin however, indicated loss of invasive properties in the plasmid cured strain. Further, pretreatment of bacterial cells with para-bromophenacyl bromide (p-BPB), a specific chemical inhibitor of phospholipase A, adversely affected adhesion to and invasion of HeLa cells in vitro, irrespective of the presence of the 220-kb plasmid indicating the possible involvement of the enzyme phospholipase A in the invasion process. Adherence of both the strains to guinea pig colonic epithelial cells (CECs) in vitro was reduced significantly on pretreatment of bacteria or CECs with p-BPB. Expression of exocellular enzymes viz. protease, elastase, phospholipase A and phospholipase C were not related to the large plasmid.

Comparative analysis of cytotoxin, hemolysin, hemagglutinin and exocellular enzymes among clinical and environmental isolates of vibrio cholerae O139 and non-O1, non-O139.

The presence of three major virulence genes toxR, tcpA and ctxA as well as expression of several putative virulence factors were compared in 12 Vibrio cholerae O139 and non-O1,non-O139 strains of clinical and environmental origin. All the strains possessed the gene encoding the regulatory protein TOXR. None of the non-O1, non-O139 strains as well as one of the O139 environmental strains carried the genes for ctxA and tcpA. Statistically significant differences in hemagglutinin and hemolysin production were observed amongst the strains depending on the source of their isolation. Expression of extracellular enzymes such as protease, elastase, neuraminidase, phospholipase A and phospholipase C, however, did not vary significantly from the groups of strains isolated from different sources.

Purification of a mannose/glucose-specific hemagglutinin/lectin from a Vibrio cholerae O1 strain.

A cell-associated mannose/glucose-specific hemagglutinin (MSHA) has been purified from a strain of Vibrio cholerae O1 by chromatography on a chitin column followed by affinity purification on Sephadex G100. The purified protein gave a single stained band of 40 kDa by SDS-PAGE, exhibited high affinity towards D-mannose and D-glucose but was resistant to L-fucose and N-acetyl-D-glucosamine. The purified MSHA was revealed as a globular form of protein under electron microscope. In immunodiffusion tests the purified MSHA produced a single precipitin band against homologous antisera and antisera raised against the whole cell bacteria without any reactivity towards antisera raised against the purified N-acetyl-D-glucosamine-specific lectin of the same bacterial strain. Immunogold labelling confirmed the location of hemagglutinin on the surface of the bacteria. Purified MSHA reacted strongly with sera from convalescent cholera patients in immunodiffusion tests.

Use of monoclonal antibodies to identify phospholipase C as the enterotoxic factor of the bifunctional hemolysin-phospholipase C molecule of Vibrio cholerae O139.

Two hybrid clones producing monoclonal antibodies (MAbs) raised against the purified enterotoxic hemolysin-phospholipase C (HlyPC) bifunctional molecule of a Vibrio cholerae O139 strain were used to study its enterotoxicity in relation to its hemolytic and enzymatic activities. Fab fragments of MAbs from ascites produced by the two hybrids neutralized the hemolytic activity of HlyPC, leaving the enzymatic activity unaffected. In ligated rabbit ileal loop and infant mouse intestine, the Fab fragments of the MAbs were not able to neutralize the enterotoxicity of HlyPC, suggesting that PC rather than Hly is the enterotoxic moiety of the molecule. The enterotoxicity of the purified PC molecule isolated from an Hly- spontaneous mutant of the HlyPC-producing parent strain further confirms this contention. The Hly molecule isolated from a PC- mutant was not diarrheagenic.

Purification and characterisation of a hemolysin with phospholipase C activity from Vibrio cholerae O139.

A hemolysin was purified from a Vibrio cholerae O139 strain which moved as a single protein band of 67 kDa in SDS-PAGE. The hemolysin showed high level of phospholipase C activity. The purified phospholipase C-hemolysin demonstrated enterotoxic activity in rabbit ileal loop, suckling mice and enhanced permeability of rabbit skin. The pI of the purified hemolysin was 6.4. Erythrocytes from rabbit, chicken, guinea pig, sheep and horse were sensitive to the purified hemolysin in decreasing order of intensity. Erythrocytes from human and cow were unaffected by purified hemolysin.

Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139.

The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the patient. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.

Purification of a cell-associated hemagglutinin from Shigella dysenteriae type 1.

A cell-associated hemagglutinin (HA) was isolated and purified from a clinical isolate of Shigella dysenteriae type 1 by affinity chromatography on a fetuin-agarose column. The purified hemagglutinin produced a single-stained protein band of around 66 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). In an immunodiffusion test, HA-antisera produced a single precipitin band against the purified HA without exhibiting any reactivity towards lipopolysaccharide (LPS) of S. dysenteriae type 1 strain. Inhibition of the hemagglutination by the glycoproteins fetuin, asialofetuin and a sugar derivative N-acetyl-neuraminic acid but not by simple sugars, suggested the specific requirement of complex carbohydrate for binding. Electron micrographs of the purified HA revealed a morphology typical of globular protein.

Role of cell-associated N-acetyl-D-glucosamine specific haemagglutinin in the adhesion of Vibrio cholerae O1 to rabbit intestinal epithelial cells in vitro.

Previously a N-acetyl-D-glucosamine specific cell-associated haemagglutinin (HA) had been purified from a Vibrio cholerae O1 strain. This study documents the role of this purified HA as an adhesin of V. cholerae O1. A significant inhibition in the adhesion of V. cholerae O1 bacterial cells to isolated rabbit intestinal brush borders (RIBB) was observed when the latter were pretreated with purified HA in ELISA. Antibody raised against purified HA and Fab (IgG) fragment of this serum inhibited adhesion of the bacteria to isolated rabbit intestinal epithelial cells (RIEC). V. cholerae O1 (both Ogawa and Inaba serovars) showed less adherence to isolated RIEC of animals immunised with the purified HA. Patients convalescing from V. cholerae O1 infection showed high ELISA titres against the purified HA indicating that it is expressed in the host during the disease process.

Antibody mediated inhibition of adhesion of Shigella dysenteriae 1 to guinea pig colonic epithelial cells in vitro.

Immunoglobulin G (IgG) fractions prepared from sera of Shigella dysenteriae 1 infected patients during the acute and convalescent phases of illness were found to be effective inhibitors of adhesion of S. dysenteriae cells to guinea pig colonic epithelial cells in vitro. The adhesion could also be inhibited by whole sera, their IgG fractions, and Fab fractions prepared from sera of rabbits immunized with whole bacteria, the outer membrane (OM) and lipopolysaccharide. The adhesive capability was best inhibited by the Fab fragment of antisera of rabbits immunized with whole bacteria.

Haemagglutinating property & cell surface hydrophobicity of Vibrio cholerae 0139.

Cell-associated haemagglutinating activity was detected in all the epidemic strains of V. cholerae 0139 and 01, isolated in different parts of the country, with erythrocytes from rabbit, rat, chicken and guinea pig. Sheep erythrocytes were unresponsive to both groups of strains. While D-mannose, alpha-methyl-D-mannoside, glucosamine, N-acetyl-D-glucosamine, thyroglobuline were effective inhibitors of the haemagglutinating activity of the V. cholerae 0139 and 01 strains, galactose and N-acetyl-D-galactosamine sensitive haemagglutinins (HAs) were detected in the V. cholerae 01 but not in the V. cholerae 0139 strains. The best medium for expression of HAs in V. cholerae 01 and 0139 strains were observed to be penassay broth and tryptic soy broth respectively. The expression of HA in V. cholerae 01 was stimulated by Ca++ and Fe in the growth medium unlike that of V. cholerae 0139 which remained unaffected by these ions.

Studies on adhesion, haemagglutination and other biological properties of Vibrio cholerae O139.

The adhesive capabilities of eight Vibrio cholerae O139 epidemic strains to isolated rabbit intestinal epithelial cells (RIEC) were observed to be high similar to those observed with a Vibrio cholerae O1 strain isolated from patients. Toxin production by the strains, measured by accumulation of fluid in rabbit ileal loop model, was high and the toxin was lethal as the animal expired within 6 h. Culture filtrates of the strains exhibited the presence of vascular permeability factor which produce induration and necrosis in the adult rabbit and guinea pig skin. All the strains showed high to moderate haemagglutinin titres against chicken erythrocytes and produced El Tor-like haemolysin. SDS-PAGE of the outer membrane preparation of the strains showed the presence of major protein component at 38 kDa region. The lethality of the toxin, high adhesive activity, shifting of the major outer membrane protein band and production of thermolabile haemolysin on Wagatsuma agar were the major variations of these epidemic strains from V. cholerae O1 and V. cholerae non-O1 strains isolated previously.

N-acetyl-D-glucosamine-specific lectin purified from Vibrio cholerae 01.

An N-acetyl-D-glucosamine-specific cell associated hemagglutinin (HA) was isolated and purified from a strain of Vibrio cholerae 01 by chitin affinity chromatography followed by separation on Bio Gel P-150. A single stained protein band of 47 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was observed with the purified HA. HA-antisera produced a single precipitin band against the purified HA in an immunodiffusion test without exhibiting any reactivity towards purified lipopolysaccharide (LPS). Purified HA, used as solid-phase antigen in an enzyme-linked immunosorbent assay (ELISA), reacted strongly with HA-antisera but cross-reacted negligibly with antisera raised against purified LPS. Hemagglutinating activity of the purified HA was highly sensitive to N-acetyl-D-glucosamine. The immunogold-labelling method using HA-antisera confirmed the location of the HA on the surface of the bacterial cells. The HA-antisera reacted with a protein component of the homologous outer membrane preparation. A significant inhibition was observed in the adhesive capability of the V. cholerae 01 strain to isolated rabbit intestinal epithelial cells (RIEC) in vitro when the later were pre-treated with the purified HA.

Adhesion of Shigella dysenteriae type 1 and Shigella flexneri to guinea-pig colonic epithelial cells in vitro.

Adhesion of bacteria to guinea-pig colonic epithelial cells in vitro was inhibited by fucose with all the four strains tested (two of Shigella dysenteriae type 1 and two of S. flexneri). N-acetyl neuraminic acid and N-acetyl mannosamine also caused inhibition, suggesting a multiplicity of receptors on the epithelial cell. Congo-red binding of the strains correlated with their adhesive ability, whereas haemagglutination of rabbit erythrocytes by the bacteria did not.

Factors affecting the colonization of isolated rabbit intestinal epithelial cells by Vibrio cholerae 01 in vitro.

The adhesive capability of Vibrio cholerae 01 strains to isolated rabbit intestinal epithelial cells was maximally expressed when the bacteria were grown in synthetic broth and was enhanced by the presence of Ca2+ in the growth media. N-Acetyl-D-glucosamine could inhibit the adhesion of the bacteria to rabbit intestinal epithelial cells as could lipopolysaccharide O-antigen from Vibrio cholerae 01 and lectin from Triticum vulgaris. Since the lipopolysaccharide is known to contain N-acetyl-D-glucosamine and because the lectin from Triticum vulgaris shows specificity for this sugar, it is probable that N-acetyl-D-glucosamine is actively involved in the adhesion of Vibrio cholerae 01 to isolated rabbit intestinal epithelial cells.

Immunochemical properties of the lipopolysaccharide O-antigen of Vibrio cholerae O1 in relation to its chemical structure.

D-Glucuronic acid and D-glucosamine have an immunodominant role in the lipopolysaccharide (LPS) O-antigen of both the Ogawa and the Inaba subtypes of Vibrio cholerae O1. This was evident from the pronounced inhibitory effect on the LPS precipitin reaction demonstrated by these monosaccharides and by oligosaccharides containing either of them which were isolated from LPS hydrolysate. There was a considerable decrease in the antibody-combining capacity of chemically modified LPS in which the carboxyl group of the glucuronic acid had been reduced. Similarly, on deamination, the O-specific polysaccharide fraction of the LPS molecule from both subtypes completely lost the ability to precipitate the LPS antibody.

Studies on the partial structure of the O-antigen of Vibrio cholera, Inaba 569 B.

Five oligosaccharides were isolated in pure state from the lipopolysaccharide of Vibrio cholera, Inaba 569 B, the their structures were elucidated. More-detailed information regarding the partial structure of the lipopolysaccharide, containing glucose, mannose, glucuronic acid, 2-amino-2-deoxyglucose, D-glycero-L-mannoheptose, and D-glycero-L-gluco-heptose, was obtained through Smith degradation, chromium trioxide oxidation, and graded hydrolysis studies of the lipopolysaccharide and its derived products.

Structural investigations of the lipopolysaccharide isolated from Vibrio cholera, Inaba 569 B.

On hydrolysis, the purified lipopolysaccharide (LPS) isolated from Vibrio cholera, Inaba 569 B, yielded glucose, mannose, a heptose behaving like D-glycero-L-manno-heptose and one behaving like D-glycero-L-gluco-heptose, 2-amino-2-deoxyglucose, and glucuronic acid in the molar ratios of approximately 9:4:5:1:2:5. Studies on the LPS, the polysaccharide (PS), and carboxyl-reduced LPS showed that the PS has a branched structure, with (1 leads to 2)-linked mannopyranosyl and a heptopyranosyl, and (1 leads to 4)-linked glucopyranosyluronic and 2-amino-2-deoxyglucopyranosyl residues in the interior part of the molecule, and glucopyranosyl and heptopyranosyl residues as nonreducing end-groups.