Purification and characterization of the soluble hemagglutinin (cholera lectin)( produced by Vibrio cholerae

Abstract

The soluble hemagglutinin (HA) (cholera lectin) produced by Vibrio cholerae strain CA401 was purified to apparent homogeneity by a sequence of ammonium sulfate fractionation, gel filtration, and preparative isoelectric focusing. Soluble HA activity was found to focus at three different pls, 6.3, 5.3, and 4.7. Each of the factors migrated as a large-molecular-weight protein in sodium dodecyl sulfatepolyacrylamide gel electrophoresis under normal conditions, and each, upon heating in sodium dodecyl sulfate was found to dissociate into 32,000-molecularweight subunits. Treating the samples with a reducing agent did not affect their mobility. Each gave a reaction of immunological identity with antiserum prepared against the others. Thus, there are apparently three distinct pH isotypes of soluble HA which exist as noncovalently associated polymers of 32,000-molecular-weight subunits. Electron microscopy of purified preparations revealed long filamentous polymers. The molecule does not stain as a glycoprotein; it is hydrophobic; it is inactivated during incubation at 25, 37, or 60'C; and it has significant protease activity. The protease activity likewise focused at pH values of 6.3 and 5.3 to 4.7, and it was inhibited by antiserum against the HA. However, whereas the HA is active at 4TC, the protease is not. The soluble HA is, therefore, a bifunctional molecule capable of mediating hemagglutination and proteolysis. Its amino acid composition is reported. Fab fragments of antibody against the purified HA inhibited attachment of heterologous serotype-biotype V. cholerae to infant rabbit small bowel. MATERIALS AND METHODS Bacterial strains. V. cholerae strain CA401 is a classical biotype, Inaba serotype originally isolated in Calcutta in 1953 by C. E. Lankford. It and other V. cholerae strains used have been described previously (4, 7, 9). HA assay. The techniques for microtiter quantitation of HA activity were as described (7). Responsive and nonresponsive chicken erythrocytes (7) used for monitoring HA activity throughout the purification scheme, were obtained from individual chickens from an inbred line of White Leghorn chickens at the University of Missouri Department of Poultry Husbandry. Purification of soluble HA. V. cholerae CA401 was grown confluently on four petri plates of meat extract agar overnight at 37°C. Growth was harvested with sterile tryptic soy broth (TSB; Difco Laboratories, Detroit, Mich.) and inoculated into 9.5 liters of TSB in each of four fermentor jars. Fermentors (New Brunswick Scientific, Inc., Edison, N.J.), to which were added 3 ml of antifoam (antifoam B,

Vibriosp. as a potentially important member of the Black Band Disease (BBD) consortium inFaviasp. corals

“…Bacterial proteolytic activity is known to be a key virulent characteristic of pathogenic Vibrio sp. (Filkelstein & Hanne, 1982). In this study, we used the qualitative casein test and the semi-quantitative azocasein test to assess the proteolytic activities of our strains.…”

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

“…Thus, like V. cholerae 01 toxin (CT) [lo], haemolysin [7], chicken erythrocyte agglutination [7], and is positive for gene sequences for zonula occludens toxin (Zot), accessory cholera enterotoxin (Ace), the core encoded pilin (Cep), and toxin coregulated pili (TcpA) [ll], and is negative for the heat-stable enterotoxin of V. cholerae non-01 (NAG-ST) [Il. Another important virulence factor of V. cholerue 01 is a haemaggulutinin/protease (Vc-HA/P) [12] which is indistinguishable from the HA/P produced by V. cholerae non-01 (NAG-HA/P) [13]. This virulence factor is thought to be involved in the pathogenesis of cholera by mediating the intestinal attachment and detachment of the organism, activating CT and other virulence factors n41.…”

Characterization of non-membrane-damaging cytotoxin of non-toxigenic Vibrio cholerae O1 and its relevance to disease

“…The ¢rst 15 amino-terminal amino acids of the puri¢ed protein was determined as Ala Gln Ala Thr Gly Thr Gly Pro Gly Gly Asn Gln Lys Thr Gly. When compared with the SWISS PROT and NBRF-PIR protein sequence libraries, the N-terminal 15 amino acid residues of O1, NMDCY showed complete homology with the N-terminal 15 residues of the soluble hemagglutinin protease (HA/P) of V. cholerae O1 ¢rst puri¢ed by Finkelstein and Hanne [20,21]. However, unlike HA/P, NMDCY puri¢ed from strain WO5 did not show HA activity at a concentration of 10 Wg using Balb/c mice and chicken erythrocytes, although the protein had signi¢cant protease activity which was inhibited by metal chelators, such as EGTA (5 mM) (Fig.…”