Vibrio anguillarum colonization of rainbow trout integument requires a DNA locus involved in exopolysaccharide transport and biosynthesis

Vibrio anguillarum, part of the normal flora of the aquatic milieu, causes a fatal haemorrhagic septicaemia in marine fish. In this study, a rainbow trout model was used to characterize the colonization of fish skin by V. anguillarum. Within 5 h after infection, the bacterium penetrated the skin mucosal layer, attached to the scales within 12 h, and formed a biofilm by 24-48 h. Two divergently transcribed putative operons, orf1-wbfD-wbfC-wbfB and wza-wzb-wzc, were shown to play a role in skin colonization and virulence. The first operon encodes proteins of unknown function. The wza-wzb-wzc genes encode a secretin, tyrosine kinase and tyrosine phosphatase, respectively, which are similar to proteins in polysaccharide transport complexes. Compared with the wild type, polar mutations in wza, orf1 and wbfD caused a decrease in exopolysaccharide biosynthesis but not lipopolysaccharide biosynthesis. The wza and orf1 mutants did not attach to fish scales; whereas, the wbfD mutant had a wild-type phenotype. Moreover, the wza and orf1 mutants had decreased exoprotease activity, in particular the extracellular metalloprotease EmpA, as well as mucinase activity suggesting that these mutations also affect exoenzyme secretion. Thus, the exopolysaccharide transport system in V. anguillarum is required for attachment to fish skin, possibly preventing mechanical removal of bacteria via natural sloughing of mucus.