Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum

Abstract

Genetic evidence has previously suggested that a zinc metalloprotease is involved in the invasive mechanism of the fish pathogen Vibrno anguMlalum NB10. In this study, the metalloprotease gene was cloned and sequenced. The sequence encodes a polypeptide (611 amino acids) that contains a putative signal sequence followed by a large leader sequence and the mature protein (44.6 kDa). Since the purified protein has a molecular mass of 36 kDa instead of the predicted 44.6 kDa, the mature protein is most likely processed a third time. Comparative analyses of the protein sequence showed high homologies to other bacterial metailoproteases within the zinc-binding and active-site regions. The Vibrio cholrae hemagglutinin/protease and the Pseudomonas aeruginosa elastase were exceptions in that the homology extended throughout the entire putative preproprotein. A chromosomal metalioprotease mutant was made via the integration of foreign DNA into the protease gene. This mutant did not secrete the metalloprotease, as determined by sodium dodecyl sulfate (SDS)-polyacrylamide protein analysis and by growth on gelatin agar. Transcomplemention of the chromosomal mutation revived the secretion of the metalloprotease and its activity on gelatin agar. Interestingly, when supernatant proteins were analyzed by gelatin-SDS-polyacrylamide electrophoresis, two different proteases (75 and 30 kDa) were detected in the mutant strain but not in the transcomplemented strain or the wild-type strain.Moreover, fish infection studies were done, and implications for the role of the metalloprotease in the virulence mechanism of V. anguiilanrm are discussed.

Vibrio vulnificus Vulnibactin, But Not Metalloprotease VvpE, Is Essentially Required for Iron-Uptake from Human Holotransferrin

“…Insertional inactivation of the vis gene was achieved using a suicide vector pNQ705. 30) A DNA fragment lacking the 5Ј and 3Ј ends of the venB gene was amplified by PCR using Vis-1 and Vis-2 primers. The amplified fragment was then cloned into pCR ® 2.1-TOPO ® vector (pCMM230), and the XbaI-HindIII fragment from pCMM230 was cloned into a suicide vector pNQ705 (pCMM232).…”

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

“…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). Similarly, a metalloprotease mutant of Vibrio vulnificus was found to express a serine protease in response that demonstrated comparable levels of proteolytic activity to the wild-type strain (Wang et al, 2008).…”

Evaluation of the vaccine potential of a cytotoxic protease and a protective immunogen from a pathogenic Vibrio harveyi strain

“…For example, the extracellular metalloprotease PrtV of Vibrio cholerae is required for host-killing [46], and the zinc metalloprotease EmpA of Vibrio anguillarum is a virulence factor that participates in pathogen-host interactions [47][48][49][50]. Both PrtV and EmpA are known to be cytotoxic to cultured host cells [51,52].…”