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Infect Immun. 1993 February; 61(2): 371-377

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

School of Pharmacy and Medical Sciences, University of South Australia, Adelaide.

ABSTRACT

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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