Isolation of carbohydrate-reactive outer membrane proteins of Aeromonas hydrophila.

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Outer membrane proteins of Aeromonas hydrophila A6 were isolated by affinity chromatography on the basis of their reactivity with trisaccharide structures analogous to the terminal trisaccharide of the H antigen of the human ABO(H) blood group system and were characterized by using antisera raised against the isolate. The outer membrane extract for affinity chromatography was prepared from pressure-disrupted outer membranes by differential centrifugation, followed by solubilization of outer membrane components in a nondenaturing, nonionic detergent. Carbohydrate-reactive outer membrane proteins (CROMPs) were then purified by affinity chromatography on two different affinity matrices composed of trisaccharides resembling the terminal trisaccharide of the H antigen, attached to inert silica beads. The relative efficiencies of H type 1 and 2 terminal trisaccharides as affinity adsorbents were established. Reactive proteins were eluted under alkaline conditions (pH 11.0) and in the presence of soluble H substance prepared from group O secretor saliva, but not by 60 mM alpha-L-fucose or under acid conditions (pH 3.0). The eluate contained at least three components (M(r)s, 43,000, 40,000, and < 14,000), as detected by immunoblot analysis with a polyvalent, polyspecific rabbit antiserum to A. hydrophila A6 (serum 3/83). A specific antiserum (serum 3/91) prepared in a rabbit by repeated immunizations with nitrocellulose containing the 43,000-Da band reacted with three bands (M(r)s, 43,000, 40,000, and < 14,000) in immunoblot analysis of solubilized outer membranes of A. hydrophila A6, suggesting that the 40,000- and < 14,000-Da elements are immunologically related to components of the 43,000-Da protein. Furthermore, pretreatment of A. hydrophila A6 with serum 3/91 reduced the strength of bacterial hemagglutination. The purified CROMPs did not agglutinate human group O erythrocytes. The reactivity of isolated CROMPs with a second CROMP-specific antibody (lipopolysaccharide-absorbed serum 3/83) was investigated. CROMPs, proteinase K-treated CROMPs, and bovine serum albumin were bound to latex beads and reacted with lipopolysaccharide-absorbed serum 3/83. Antibodies eluted from CROMP-latex inhibited hemagglutination of human erythrocytes by A. hydrophila A6 to a titer of 4. Antibody eluted from proteinase K-treated CROMP-latex beads showed hemagglutination inhibition activity only when undiluted. There was no hemagglutination inhibition antibody activity detectable in the eluate from bovine serum albumin-latex beads. These results show that antibodies which react with the isolated CROMPs also react with an H-antigen-reactive hemagglutinin of A. hydrophila A6. The possibility that CROMPs act as an adhesin, or adhesins, and contribute to the virulence of this organism is discussed.

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