Metalloprotease vsm is the major determinant of toxicity for extracellular products of Vibrio splendidus.
Genomic data combined with reverse genetic approaches have contributed to the characterization of major virulence factors of Vibrio species; however, these studies have targeted primarily human pathogens. Here, we investigate virulence factors in the oyster pathogen Vibrio splendidus LGP32 and show that toxicity is correlated to the presence of a metalloprotease and its corresponding vsm gene. Comparative genomics showed that an avirulent strain closely related to LGP32 lacked the metalloprotease. The toxicity of LGP32 metalloprotease was confirmed by exposing mollusk and mouse fibroblastic cell lines to extracellular products (ECPs) of the wild type (wt) and a vsm deletion mutant (Deltavsm mutant). The ECPs of the wt induced a strong cytopathic effect whose severity was cell type dependent, while those of the Deltavsm mutant were much less toxic, and exposure to purified protein demonstrated the direct toxicity of the Vsm metalloprotease. Finally, to investigate Vsm molecular targets, a proteomic analysis of the ECPs of both LGP32 and the Deltavsm mutant was performed, revealing a number of differentially expressed and/or processed proteins. One of these, the VSA1062 metalloprotease, was found to have significant identity to the immune inhibitor A precursor, a virulence factor of Bacillus thuringiensis. Deletion mutants corresponding to several of the major proteins were constructed by allelic exchange, and the ECPs of these mutants proved to be toxic to both cell cultures and animals. Taken together, these data demonstrate that Vsm is the major toxicity factor in the ECPs of V. splendidus.
Institut Pasteur, Unité Plasticité du Génome Bactérien, CNRS URA 2171, Paris, France.
Johan
J
Binesse
Claude
C
Delsert
Denis
D
Saulnier
Marie-Christine
MC
Champomier-Vergès
Monique
M
Zagorec
Hélène
H
Munier-Lehmann
Didier
D
Mazel
Frédérique
F
Le Roux
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000633:toxicity*; D002478:Cells, Cultured; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005347:Fibroblasts / Q000187:drug effects; D017353:Gene Deletion; D023281:Genomics; D045726:Metalloproteases / Q000235:genetics / Q000633:toxicity*; D051379:Mice; D008969:Molecular Sequence Data; D008974:Mollusca / Q000187:drug effects; D020543:Proteome / Q000032:analysis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000201:enzymology*; D037521:Virulence Factors / Q000235:genetics / Q000633:toxicity*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D020543:Proteome; D037521:Virulence Factors; D045726:Metalloproteases