The Vibriolysin-Like Protease VnpA and the Collagenase ColA Are Required for Full Virulence of the Bivalve Mollusks Pathogen Vibrio neptunius

Abstract

Vibrio neptunius is an important pathogen of bivalve mollusks worldwide. Several metalloproteases have been described as virulence factors in species of Vibrio that are pathogenic to bivalves, but little is known about the contribution of these potential virulence factors to Vibrio neptunius pathogenesis. In silico analysis of the genome of V. neptunius strain PP-145.98 led to the identification of two hitherto uncharacterized chromosomal loci encoding a probable vibriolysin-like metalloprotease and a putative collagenase, which were designated VnpA and ColA, respectively. Single defective mutants of each gene were obtained in V. neptunius PP-145.98, and the phospholipase, esterase and collagenase activities were studied and compared with those of the wild-type strain. The results showed that the single inactivation of vnpA resulted in a 3-fold reduction in phospholipase/esterase activity. Inactivation of colA reduced the collagenase activity by 50%. Finally, infection challenges performed in oyster larvae showed that ΔvnpA and ΔcolA-single mutant strains of V. neptunius-are between 2-3-fold less virulent than the wild-type strain. Thus, the present work demonstrates that the production of both VnpA and ColA is required for the full virulence of the bivalve pathogen V. neptunius.

Keywords: ColA; Vibrio neptunius; VnpA; bivalve mollusk pathogen; collagenase; metalloprotease; virulence.

Figure 1

Conserved domains and motifs found in VnpA and ColA metalloproteases.

Figure 2

Representative results of phospholipase, esterase, gelatinase and hemolytic activities in the PP-145.98 wild-type strain and in its derivatives vnpA- and colA-defective mutants. Numbers denote halo sizes in millimeters.

Figure 3

Survival curves after challenge in oyster larvae (Ostrea edulis) with the wild-type V. neptunius strain or ΔcolA- or ΔvnpA-defective mutants. Asterisks denote statistically significant differences between strains (*** p < 0.001).