title: First description of French V. tubiashii strains pathogenic to mollusk: II. Characterization of properties of the proteolytic fraction of extracellular products.
abstract: Extracellular products (ECPs) of the French Vibrio tubiashii strain 07/118 T2 were previously reported to be toxic for the Pacific oyster Crassostrea gigas. In this study we now assessed host cellular immune responses and bacterial potential effectors by which these ECPs can be associated with host damages. The adhesion capacity (28% inhibition) and phagocytosis ability (56% inhibition) of oyster hemocytes were the main functions affected following in vitro contact between hemocytes and V. tubiashii ECPs. This may be linked to the demonstration of the capability of ECPs to cleave various cellular substrates as oyster collagen. Moreover, a strong metalloproteolytic activity was recorded with general (azocasein) and specific (ADAM) substrates and characterized by the use of standard inhibitors and metal ions. The addition of 1,10-phenanthroline and Zn2+ decreased proteolytic activity by about 80% and 50% respectively, confirming the presence of zinc metalloproteolytic activity in the ECPs. Mass spectrometry analyses of crude ECPs identified an extracellular zinc metalloprotease encoded by a gene with an open reading frame of 1821 bp (606 aa). Consensus zinc-binding motifs specific to thermolysin family and some glycosylation and phosphorylation sites were located on the deduced protein sequence. Taken together, our results suggest that this (these) zinc metalloprotease(s) might contribute to the impairment of hemocyte immunological functions; however, their direct involvement in ECPs toxicity remains to be demonstrated.
authors.affiliation: UMR 7266 CNRS-Université de La Rochelle, LIENSs, Equipe Approches Moléculaires, Environnement-Santé, Avenue Michel Crépeau, 17000 La Rochelle, France; Ifremer, SG2M-LGPMM, Laboratoire de Génétique et Pathologie des Mollusques Marins, Avenue de Mus de Loup, 17390 La Tremblade, France; Fédération de Recherche en Environnement et Développement Durable, FR CNRS 3097, Université de La Rochelle, France.
authors.forename: Rachida
authors.initials: R
authors.lastname: Mersni-Achour
authors.affiliation: UMR 7266 CNRS-Université de La Rochelle, LIENSs, Equipe Animaux MARins à la variabilité Environnementale, 2 rue Olympe de Gouges, 17000 La Rochelle, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Imbert-Auvray
authors.affiliation: UMR 7266 CNRS-Université de La Rochelle, 2 rue Olympe de Gouges, 17000 La Rochelle, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Huet
authors.affiliation: UMR 7266 CNRS-Université de La Rochelle, LIENSs, Equipe Approches Moléculaires, Environnement-Santé, Avenue Michel Crépeau, 17000 La Rochelle, France.
authors.forename: Yosra
authors.initials: Y
authors.lastname: Ben Cheikh
authors.affiliation: Ifremer, SG2M-LGPMM, Laboratoire de Génétique et Pathologie des Mollusques Marins, Avenue de Mus de Loup, 17390 La Tremblade, France.
authors.forename: Nicole
authors.initials: N
authors.lastname: Faury
authors.affiliation: UMR 7266 CNRS-Université de La Rochelle, LIENSs, Equipe Approches Moléculaires, Environnement-Santé, Avenue Michel Crépeau, 17000 La Rochelle, France.
authors.forename: Ibtissem
authors.initials: I
authors.lastname: Doghri
authors.affiliation: UMR 7266 CNRS-Université de La Rochelle, LIENSs, Equipe Animaux MARins à la variabilité Environnementale, 2 rue Olympe de Gouges, 17000 La Rochelle, France.
authors.forename: Sonia
authors.initials: S
authors.lastname: Rouatbi
authors.affiliation: UMR 7266 CNRS-Université de La Rochelle, LIENSs, Equipe Approches Moléculaires, Environnement-Santé, Avenue Michel Crépeau, 17000 La Rochelle, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Bordenave
authors.affiliation: Ifremer, SG2M-LGPMM, Laboratoire de Génétique et Pathologie des Mollusques Marins, Avenue de Mus de Loup, 17390 La Tremblade, France.
authors.forename: Marie-Agnès
authors.initials: MA
authors.lastname: Travers
authors.affiliation: Ifremer, SG2M-LGPMM, Laboratoire de Génétique et Pathologie des Mollusques Marins, Avenue de Mus de Loup, 17390 La Tremblade, France; Ifremer, Centre Ifremer du Pacifique, UMR 241 Ecosystèmes Insulaires Océaniens, Tahiti, 98719 Taravao, French Polynesia.
authors.forename: Denis
authors.initials: D
authors.lastname: Saulnier
authors.affiliation: UMR 7266 CNRS-Université de La Rochelle, LIENSs, Equipe Approches Moléculaires, Environnement-Santé, Avenue Michel Crépeau, 17000 La Rochelle, France; Fédération de Recherche en Environnement et Développement Durable, FR CNRS 3097, Université de La Rochelle, France. Electronic address: ingrid.fruitier@univ-lr.fr.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Fruitier-Arnaudin
mesh_terms: D000818:Animals; D049896:Crassostrea / Q000382:microbiology*; D005110:Extracellular Space / Q000201:enzymology; D013058:Mass Spectrometry; D045726:Metalloproteases / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014733:Vibrio / Q000235:genetics* / Q000472:pathogenicity*
chemical_list: D045726:Metalloproteases
keywords: ADAM substrate; ECPs; Mass spectroscopy; Oyster immune responses; Thermolysin-metalloprotease; Vibrio