Vibrio aestuarianus zinc metalloprotease causes lethality in the Pacific oyster Crassostrea gigas and impairs the host cellular immune defenses.
Extracellular products (ECPs) of the pathogenic Vibrio aestuarianus 01/32 were previously reported to display lethality in Crassostrea gigas oysters and to cause morphological changes and immunosuppression in oyster hemocytes. To identify the source of this toxicity, biochemical and genetic approaches were developed. ECP protease activity and lethality were shown to be significantly reduced following incubation with metal chelators, suggesting the involvement of a zinc metalloprotease. An open reading frame of 1836 bp encoding a 611-aa metalloprotease (designated Vam) was identified. The deduced protein sequence showed high homology to other Vibrio metalloproteases reported to be involved in pathogenicity. To further confirm the role of this enzyme in ECP toxicity, a plasmid carrying the vam gene under the control of an araC-P(BAD) expression cassette was transferred to a Vibrio splendidus related strain, LMG20012(T), previously characterized as non-pathogenic to oysters. Expression of Vam conferred a toxic phenotype to LMG20012(T) ECPs in vivo and cytotoxicity to oyster hemocytes in vitro. Collectively, these data suggest that the Vam metalloprotease is a major contributor to the toxicity induced by V. aestuarianus ECPs and is involved in the impairment of oyster hemocyte functions.
Labreuche
Yannick
Y
UMR 100 IFREMER Physiologie et Ecophysiologie des Mollusques Marins, Centre de Brest, B.P. 70, 29280 Plouzané, France. yannick.labreuche@ifremer.fr
Le Roux
Frédérique
F
Henry
Joël
J
Zatylny
Céline
C
Huvet
Arnaud
A
Lambert
Christophe
C
Soudant
Philippe
P
Mazel
Didier
D
Nicolas
Jean-Louis
JL
D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004768:Enterotoxins / Q000633:toxicity*; D005110:Extracellular Space / Q000737:chemistry; D007111:Immunity, Cellular / Q000187:drug effects*; D008666:Metalloendopeptidases / Q000633:toxicity*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010125:Ostreidae / Q000187:drug effects* / Q000235:genetics* / Q000276:immunology; D017422:Sequence Analysis, DNA; D014733:Vibrio / Q000201:enzymology*
D017931:DNA Primers; D004768:Enterotoxins; D008666:Metalloendopeptidases