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Applied and Environmental Microbiology, December 2008, p. 7108-7117, Vol. 74, No. 23
0099-2240/08/$08.00+0     doi:10.1128/AEM.01261-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products of Vibrio splendidus ^,

Johan Binesse,¹ Claude Delsert,^2,3 Denis Saulnier,² Marie-Christine Champomier-Vergès,⁴ Monique Zagorec,⁴ Hélène Munier-Lehmann,⁵ Didier Mazel,¹ and Frédérique Le Roux^1,2*

Institut Pasteur, Unité Plasticité du Génome Bactérien, CNRS URA 2171, 25 rue du Dr Roux, 75015 Paris, France,¹ Ifremer, Laboratoire de Génétique et Pathologie, BP33, 17390 La Tremblade, France,² CNRS UMR 5237, Centre de Recherche en Biochimie des Macromolécules, 1919 route de Mende, 34293 Montpellier, France,³ INRA, Unité Flore Lactique et Environnement Carné (UR309), Domaine de Vilvert, 78350 Jouy-en-Josas, France,⁴ Institut Pasteur, Unité de Chimie Organique, 25 rue du Dr Roux, 75015 Paris, France⁵

Received 6 June 2008/ Accepted 3 September 2008

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 (vsm mutant). The ECPs of the wt induced a strong cytopathic effect whose severity was cell type dependent, while those of the vsm 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 vsm 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.

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* Corresponding author. Mailing address: Institut Pasteur, Unité Plasticité du Génome Bactérien, CNRS URA 2171, 25 rue du Dr Roux, 75724 Paris, France. Phone: 33 1 40 61 32 87. Fax: 33 1 45 68 88 34. E-mail: fleroux{at}pasteur.fr

Published ahead of print on 3 October 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, December 2008, p. 7108-7117, Vol. 74, No. 23
0099-2240/08/$08.00+0     doi:10.1128/AEM.01261-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.