Purification and characterization of novel hemagglutinins from Vibrio mimicus: A 39-kilodalton major outer membrane protein and lipopolysaccharide

Abstract

Two hemagglutinins (HAs) mediating the agglutinability to rabbit erythrocytes were isolated from 32-h culture supernatant of enterotoxigenic strain E-33 of Vibrio mimicus by ultrafiltration followed by gel filtration and anion-exchange column chromatography. The HAs were designated R-HA and C- HA on the basis of specific hemagglutinating activity towards rabbit erythrocytes only (R-HA) and towards chicken and rabbit erythrocytes (C-HA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent staining with Coomassie brilliant blue revealed no detectable protein band and a single band of M(r) 39,000 in the case of R-HA and C-HA, respectively. However, silver staining of the gel containing R-HA revealed the appearance of low-molecular-weight material. These two HAs differed from each other and from previously reported HA/protease in receptor specificity, molecular composition, and biochemical and immunochemical properties. No simple sugar other than glycoproteins, including mucin, inhibited hemagglutinating activities of both C-HA and R-HA. Rabbit antibody against R-HA or C-HA could agglutinate E-33 whole cells, implying a possible cell surface origin of the two HAs. The isolated E-33 lipopolysaccharide (LPS) or its polysaccharide moiety conferred biochemical and immunochemical properties identical to those of R-HA, confirming that the R-HA represents polysaccharide of LPS. The LPS preparations from heterologous strains of Vibrio mimicus and Vibrio cholerae non-O1 confirmed that the hemagglutinating ability is a common function of LPS. On the other hand, the antibody against C-HA specifically recognized a major outer membrane protein (OMP) with an M(r) of around 39,000 in both homologous and heterologous strains of V. mimicus, suggesting an OMP origin of C-HA. Furthermore, the antibody recognized a major OMP with an M(r) of around 37,000 in V. cholerae. Although the immunogenicity of LPS and OMP is well documented for important intestinal pathogens, the hemagglutinating properties of such attractive cell surface components are hitherto unrecognized and will definitely contribute towards understanding their role in bacterial adherence.