Four novel hemolysin genes of Vibrio anguillarum and their virulence to rainbow trout

Four nucleotide sequences showing homology to known hemolysin genes were cloned and sequenced from V. anguillarum strain H775-3. The four genes, vah2, vah3, vah4 and vah5, have open reading frames encoding polypeptides of 291, 690, 200 and 585 amino acid residues, respectively, with predicted molecular masses of 33, 75, 22 and 66 KDa, respectively. VAH2 is most closely related to a putative hemolysin of Vibrio vulnificus YJ016 (89% identity). VAH3 is most closely related to a hemolysin-related protein in Vibrio cholerae O1 (68% identity). VAH4 is most closely related to a thermostable hemolysin in V. cholerae O1 (72% identity). VAH5 is most closely related to a putative hemolysin in V. cholerae O1 (73% identity). The purified hemolysin proteins showed hemolytic activities against erythrocyte of fish, sheep and rabbit. Four strains of V. anguillarum mutants were constructed, each deficient in one of the hemolysin genes. Each mutant was less virulent than V. anguillarum H775-3 to juvenile rainbow trout (Oncorhynchus mykiss), indicating that each hemolysin gene contributes to the virulence of V. anguillarum H775-3.