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.

Bacterial diseases in marine bivalves

“…Considering the potential impact of this bacterium, draft genomes (Temperton et al, 2011) and a complete genome (Richards et al, 2014b) were recently sequenced and will enhance the exploration of potential virulence factors. The importance of cytolysin, outer membrane proteins and secreted metalloproteases has already been described for different V. tubiashii strains and constitute a bacterial virulence repertoire (Kothary et al, 2001;Delston et al, 2003;Beaubrun et al, 2008;Hasegawa et al, 2008;Mersni Achour et al, 2014). As in many diseases, environmental conditions can influence the interaction of V. tubiashii with its host.…”

Factors other than metalloprotease are required for full virulence of French Vibrio tubiashii isolates in oyster larvae

“…Demonstration of their virulence was carried out and toxicity of their ECPs was confirmed by experimental challenges with juvenile Pacific oysters. Moreover, Mersni-Achour et al (2014) demonstrated that the ECPs of 07/118 T2, a representative strain of the group, inhibited the adhesion capacity and phagocytosis activity of C. gigas haemocytes. Complementary biochemical analyses showed that the proteolytic fraction of ECPs contained active and thermostable extracellular zinc metalloprotease(s).…”

New Insights into Pathogenic Vibrios Affecting Bivalves in Hatcheries: Present and Future Prospects

“…In relation to the virulence factors, Mersni-Achour et al (2014) identified an extracellular zinc metalloprotease belonging to the thermolysin family, close to predicted extracellular zinc metalloproteases of V. tubiashii . Later, Mersni-Achour et al (2015) studied the two major fractions (F1 and F2) from ECPs by GP-HPLC and found differences in their toxicity to larvae (43% mortality for F1 fraction, 70% in the presence of the F2 fraction and 100% mixing both fractions).…”

“…Strains described in the initial study were isolated from O. edulis and R. philippinarum during disease outbreaks in different Spanish hatcheries (Prado et al, 2005, 2015). Interestingly, Dubert et al (2016e) included in the description of the new taxa a French isolate initially identified as V. tubiashii , and highly pathogenic for C. gigas larvae and spat (Mersni-Achour et al, 2014, 2015; Travers et al, 2014). Recently, Dubert et al (2017) have reported a mortality event in larvae of other clam species, namely R. decussatus , involving this bacterial species.…”