Mutational analysis of the zinc metalloprotease EmpA ofVibrio anguillarum

Abstract

The extracellular zinc metalloprotease, EmpA, is a putative virulence factor involved in pathogenicity of the fish pathogen Vibrio anguillarum. The 611-amino acid precursor of this enzyme is encoded by the empA gene. The residues His346, His350, Glu370, Glu347, His429, Tyr361 and Asp417 are highly conserved and putatively function together at the active site of the enzyme. In this study, empA was inserted into pET24d(+) and expressed in Escherichia coli strain BL21(DE3) as a 6 x His tagged protein (r-EmpA). All the conserved residues of EmpA mentioned above were individually mutated by site-directed mutagenesis and the mutants were also expressed (m-r-EmpAs). r-EmpA and m-r-EmpAs were purified, and assayed for their proteolytic activities with azocasein as the substrate and cytotoxicities on a flounder gill cell line. m-r-EmpAs that had been mutated at His346, His350, Glu370 and Glu347 almost completely lost their proteolytic activity and cytotoxicity, pointing towards the essential roles played by these residues. In contrast, those mutated at Tyr361, His429 and Asp417 still retained a partial proteolytic activity and cytotoxicity. Our results indicate that these conserved residues play important roles in enzymatic activity and that the proteolytic activity of the enzyme is involved in the pathogenesis of V. anguillarum

Identification and Characterization of Epp, the Secreted Processing Protease for the Vibrio anguillarum EmpA Metalloprotease

“…The 36-kDa peptide was detected only if protein samples were boiled prior to SDS-PAGE and Western blot analysis (29)(30)(31). Similar results were obtained using V. anguillarum W-1 supernatant (31).…”

“…Recent studies using recombinant EmpA synthesized in E. coli (29)(30)(31) have suggested that the presence of a stable ϳ36-kDa EmpA form is the result of a thermoinduced self-proteolysis of a 44.6-kDa precursor (30). The 36-kDa peptide was detected only if protein samples were boiled prior to SDS-PAGE and Western blot analysis (29)(30)(31).…”

Comparative analysis and mutation effects of fpp2–fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum

“…The extracellular proteolytic activity in cell-free supernatants was determined by azocasein assay (Secades et al, 2001) proteolytic enzymes, as reported in Vibrio cholerae, in which production is increased in hyperswarmer mutants (Gardel & Mekalanos, 1996). Extracellular proteolytic activities have been reported to be involved in the virulence of different pathogens, including a number of fish pathogenic bacteria (Fernández et al, 2003;Yang et al, 2007;Zhang et al, 2009). However, the implication of proteases in pathogenesis is not a general rule, as shown in this study in a rainbow trout model.…”

“…Although extracellular proteolytic enzymes have been considered as virulence factors in many bacterial fish pathogens, comparative analyses of wild-type strains and isogenic mutants devoid of proteolytic activity have seldom been performed. In particular, proteases from Vibrio anguillarum (Yang et al, 2007), Yersinia ruckeri (Fernández et al, 2003), Pseudomonas fluorescens (Zhang et al, 2009) and Moritella viscosa (Bjornsdottir et al, 2009) have been clearly proved to be involved in bacterial virulence in fish.…”

An Overview of Virulence-Associated Factors of Gram-Negative Fish Pathogenic Bacteria

“…When compared with wild type strain, this mutant had a 1,000-fold higher LD 50 value after immersion infection of rainbow trout. In the work of Yang et al (2007), it is also proposed that an extracellular zinc metalloprotease, EmpA, is a putative virulence factor of the fish pathogen L. anguillarum.…”