The regulation and processing of the EmpA metalloprotease of Vibrio anguillarum.

摘要

Vibrio anguillarum is the causative agent of vibriosis, a systemic disease of fish characterized by a hemorrhagic septicemia that results in high mortalities. EmpA has been identified as a virulence factor during infection of the GI tract of juvenile Atlantic salmon. EmpA activity is dependent on successful secretion and processing of the nascent protein to an active mature protease.;This study was conducted to identify and characterize the gene(s) involved in the processing mechanism of the EmpA protein. Pro-EmpA is secreted out of the cell and is activated by the cleavage of a ∼10-kDa propeptide. It has been hypothesized that an additional protease is responsible for the activation of pro-EmpA to the mature protease.;Mini-Tn10Km mutagenesis identified one protease negative mutant. The region surrounding the mutation was cloned and sequenced, revealing epp, which encodes for a 918 amino acid putative protease. Western blot analysis of the culture supernatant from the epp mutant strain revealed only the presence of the ∼46-kDa pro-EmpA form, while the wild-type contains both the ∼46-kDa proenzyme and the mature ∼36-kDa enzyme. The epp mutation was complemented restoring protease activity and EmpA processing to wild-type levels;EmpA transcription is dependent upon several gene products, such as VanT and RpoS, as well as salmon GI mucus. We measured epp transcript levels in V. anguillarum vanT and rpoS mutant strains to determine if there are any similarities to empA regulation. Epp expression was reduced > 90% in the vanT mutant strain suggesting that epp transcription is largely dependent upon VanT. The rpoS mutant strain exhibited a 2.6-fold increase in epp expression suggesting that epp is not under the direct control of RpoS. We also determined that salmon GI mucus is also a strong inducer of epp expression. Epp was found to have no effect on the virulence of V. anguillarum against juvenile Atlantic salmon when infected by i.p. injection.;Finally, we investigated the correlation between protease and hemolytic activities in the wild-type strain M93Sm. Our results strongly support the hypothesis that hemolytic activity is related to protease activity, where hemolytic activity is inhibited post-translationally by EmpA metalloprotease.