The extracellular metalloprotease of Vibrio tubiashii is a major virulence factor for Pacific oyster (Crassostrea gigas) larvae

Department of Biomedical Sciences, Oregon State University, Corvallis, OR 97331

^* To whom correspondence should be addressed. Email: hasec{at}science.oregonstate.edu.

	Abstract

Vibrio tubiashii is a recently reemerging pathogen of larval bivalve mollusks, causing both a toxigenic and invasive disease. Marine Vibrio produce an array of extracellular products as potential pathogenicity factors. Culture supernatants of V. tubiashii have been shown to be toxic to oyster larvae and were reported to contain a metalloprotease and a cytolysin/hemolysin. However, the structural genes responsible for these proteins have yet to be identified and it is uncertain which extracellular products play a role in pathogenicity. We investigated the effects of the metalloprotease and hemolysin secreted by V. tubiashii on its ability to kill Pacific oyster (Crassostrea gigas) larvae. While V. tubiashii supernatants treated with metalloprotease inhibitors severely reduced the toxicity to oyster larvae, inhibition of the hemolytic activity did not affect larval toxicity. We identified the structural genes of V. tubiashii encoding a metalloprotease (vtpA) and a hemolysin (vthA). Sequence analyses revealed that VtpA shared high homology with metalloproteases from a variety of Vibrio species, while VthA showed high homology only to the cytolysin/hemolysin of V. vulnificus. Compared to the wild type strain, a VtpA mutant of V. tubiashii not only produced reduced amounts of protease, but also showed decreased toxicity to C. gigas larvae. V. cholerae strains carrying the vtpA or the vthA genes successfully secreted the heterologous proteins. Culture supernatants of V. cholerae carrying vtpA, but not vthA, were highly toxic to Pacific oyster larvae. Together, these results suggest that the V. tubiashii extracellular metalloprotease is importantin in its pathogenicity to C. gigas larvae.