Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products ofVibrio splendidus

Abstract

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.Vibrionaceae are a predominant family of gram-negative bacteria found in aquatic environments (30). Bacteria within this family demonstrate a high degree of genetic diversity and are able to colonize very different types of niches. They live freely as planktonic forms in the water column or are associated in biofilms or with host organisms as pathogenic, commensal, or mutualistic bacteria. To date, eight genome sequences from Vibrionaceae have been made available: those of Vibrio cholerae strains N16961 and 0395, V. parahaemolyticus RIMD2210633, V. vulnificus strains YJ016 and CMCP6, V. fischeri ES114, V. harveyi ATCC BAA-1116, and Photobacterium profundum SS9 (3,10,19,24,32). More recently, we have completed the sequencing of the genome of V. splendidus strain LGP32, an oyster (Crassostrea gigas) pathogen

Changes induced by two strains of Vibrio splendidus in haemocyte subpopulations of Mya arenaria, detected by flow cytometry with LysoTracker

“…Moreover, it has been found that this strain possesses the pathogenic factor vsm, a metalloprotease recently demonstrated to be the most important toxicity factor in the extracellular products of LGP32 (Binesse et al 2008). It is likely that this pathogenic factor is associated with the significant degranulation of Mya arenaria haemocytes in the present study.…”

Genomic and proteomic analyses of the coral pathogen Vibrio coralliilyticus reveal a diverse virulence repertoire

“…Metalloproteases have several catalytic activities related to virulence but little is known about the diversity of these potential virulence factors in coral pathogenic bacteria. In the bivalve pathogen V. splendidus, the deletion of the Vsm metalloprotease resulted in the expression of other metalloproteases that were found to compensate for some of the functions fulfilled by the vsm gene product (Binesse et al, 2008). In the fish pathogen V. anguillarum, mutants lacking a functional EmpA metalloprotease expressed two different proteases that were not detected in the wild-type strain suggesting that these proteases might compensate for the loss of EmpA (Milton et al, 1992;Varina et al, 2008).…”

“…Alleles carrying an internal deletion were generated in vitro using a two-step PCR construction method (Binesse et al, 2008). Using genomic DNA of the strain P1, two independent PCR amplifications of 500 bp regions encompassing vcpA (GeneBank accession number GQ452012) were performed using primer pairs: C1: 5 0 -gcccgaattcATGAAACAACGTCAAATG CTTTGGC-3 0 and C2: 5 0 -CAAAACCTTTACGTACAT CCCAACGCAACAAAAAAGTCTACCATGTAAACG-3 0 ; or C3: 5 0 -CGTTTACATGGTAGACTTTTTTGTTGCG TTGGGATGTACGTAAAGGTTTTG-3 0 and C4: 5 0 -gcc cgaattcTTAGTCTAATCTTAGTGTCACGC-3 0 (Lower nucleotides are EcoRI enzyme sites).…”

Transcriptomic profiling of the oyster pathogen Vibrio splendidus opens a window on the evolutionary dynamics of the small RNA repertoire in the Vibrio genus

“…This Vibrio has been associated with major oyster summer mortality outbreaks over the past 15 years (Gay et al 2004a), and the strain V. splendidus LGP32 causes mortalities when injected to oysters (Gay et al 2004b;Le Roux et al 2007). The genome of V. splendidus was sequenced (Le Roux et al 2009), and several potential virulence factors were identified (Binesse et al 2008;Duperthuy et al 2010). The genome is nearly 5 Mb, 20% larger than that of V. cholerae, and V. splendidus chromosome II is 56% larger than its V. cholerae counterpart, indicating the possibility of many new sRNAs being present in this species.…”