The Extracellular Metalloprotease ofVibrio tubiashiiIs a Major Virulence Factor for Pacific Oyster (Crassostrea gigas) Larvae

Abstract

Vibrio tubiashii is a recently reemerging pathogen of larval bivalve mollusks, causing both toxigenic and invasive disease. Marine Vibrio spp. produce an array of extracellular products as potential pathogenicity factors. Culture supernatants of V. tubiashii have been shown to be toxic to oyster larvae and were reported to contain a metalloprotease and a cytolysin/hemolysin. However, the structural genes responsible for these proteins have yet to be identified, and it is uncertain which extracellular products play a role in pathogenicity. We investigated the effects of the metalloprotease and hemolysin secreted by V. tubiashii on its ability to kill Pacific oyster (Crassostrea gigas) larvae. While V. tubiashii supernatants treated with metalloprotease inhibitors severely reduced the toxicity to oyster larvae, inhibition of the hemolytic activity did not affect larval toxicity. We identified structural genes of V. tubiashii encoding a metalloprotease (vtpA) and a hemolysin (vthA). Sequence analyses revealed that VtpA shared high homology with metalloproteases from a variety of Vibrio species, while VthA showed high homology only to the cytolysin/hemolysin of Vibrio vulnificus. Compared to the wild-type strain, a VtpA mutant of V. tubiashii not only produced reduced amounts of protease but also showed decreased toxicity to C. gigas larvae. Vibrio cholerae strains carrying the vtpA or vthA gene successfully secreted the heterologous protein. Culture supernatants of V. cholerae carrying vtpA but not vthA were highly toxic to Pacific oyster larvae. Together, these results suggest that the V. tubiashii extracellular metalloprotease is important in its pathogenicity to C. gigas larvae.Vibriosis caused by marine Vibrio species is considered one of the most serious diseases of hatchery-reared oyster larvae (10,11,17,47,52). The disease is characterized by a rapid and dramatic reduction in larval motility, detached vela, and necrotic soft tissue, which lead to high mortality rates, exceeding 90% within 1 day of infection (45). Pathogenic agents that cause larval bivalve vibriosis have intermittently and severely curtailed shellfish hatchery production on the Atlantic and Pacific coasts of the United States, causing substantial losses in the industry (3, 10, 13). Vibrio tubiashii, a bacterial species first reported by Tubiash et al. (51), was identified as a causative agent of vibriosis (originally referred to as bacillary necrosis) in larval and juvenile bivalves of the hard clam (Mercenaria mercenaria) and Eastern oyster (Crassostrea virginica). Estes et al. (14) characterized a number of pathogenic and nonpathogenic bacterial strains from diseased Pacific oysters (Crassostrea gigas) at shellfish hatcheries on the Pacific coast of North America and described some of the highly pathogenic bacterial isolates as V. tubiashii.The genus Vibrio is the largest member of the family Vibrionaceae, which includes gram-negative and curved rod-shaped facultative anaerobes. The genus consists of at least 30 known species, which are w...

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

“…In V. tubiashii, the zinc-metalloprotease VtpA regulates VthA hemolysin expression by means of a posttranscriptional mechanism (Hasegawa and Häse, 2009). The VtpA mutant of V. tubiashii had decreased pathogenic potential in C. gigas larvae (Hasegawa et al, 2008). The hyper expression of VthA did not increase the pathogenic potential of V. tubiashii in C. gigas.…”

“…The VcpA zinc-metalloprotease has been identified in previous studies as the key virulence factor likely important in the bacterial infection of corals and implicated in coral tissue necrosis (Ben-Haim et al, 2003a, b;Sussman et al, 2008). The V. coralliilyticus vcpA gene is orthologous to the vtpA gene from the oyster pathogen Vibrio tubiashii and similarly identified as central for strain virulence (Hasegawa et al, 2008). A DvcpA V. coralliilyticus P1 mutant demonstrated the same pathogenic potential as the wild-type strain in two animal models and a whole cell Symbiodiunium bioassay evaluated in this study, highlighting that the pathogenicity of vibrios in marine animals is a complex interplay of multiple genetic factors and unlikely the result of one determinant.…”

“…We suggest that vcpA, vcpB, and vchA are paralogous genes. VcpB and VchA (both from M4 family) may enable P1 to cause disease as this family is known to be thermolysins with high proteolytic activity in vibrios (Hasegawa et al, 2008). In addition, the collagenase metallopeptidases (U32) degrade type I collagens and help in the invasion of the host.…”

TetR-Type Transcriptional Regulator VtpR Functions as a Global Regulator in Vibrio tubiashii

“…One of the shellfish pathogenic Vibrio species, Vibrio tubiashii, is a recently reemerging pathogen of several species of bivalve larvae including oysters, clams, and geoducks (9, 10). Disease caused by V. tubiashii is characterized by a reduction in larval motility, detached vela, and necrotic soft tissue, accompanied by high mortality rates (42).Although only limited information is available about many aspects of V. tubiashii, including virulence factors produced, previous studies demonstrated that V. tubiashii strains, including RE22 and RE98, showed the highest degree of disease severity (10), with the production of high levels of extracellular metalloprotease (VtpA) and hemolysin (VthA) (7,19,25). We showed in a previous study that the purified VtpA protein caused significantly high toxicity to oyster larvae, strongly suggesting that the VtpA is a structural toxin to the host (18).…”

“…In addition, we recently observed that the purified VtpA directly inactivates VthA hemolysin (17). Although virulence of V. tubiashii is likely multifactorial, the metalloprotease VtpA appears to be the main secreted toxin in V. tubiashii supernatants (18,19).Typically, this type of metalloprotease is tightly regulated by a TetR family of DNA-binding transcriptional regulatory proteins in a variety of Vibrio species (5,22,23,35,39). For example, in V. vulnificus a deletion of the TetR family regulator, SmcR, results in severely reduced production of the VvpE metalloprotease (22).…”