Identification and characterization of Epp, the secreted processing protease for the Vibrio anguillarum EmpA metalloprotease.
The zinc metalloprotease EmpA is a virulence factor for the fish pathogen Vibrio anguillarum. Previous studies demonstrated that EmpA is secreted as a 46-kDa proenzyme that is activated extracellularly by the removal of an approximately 10-kDa propeptide. We hypothesized that a specific protease is responsible for processing secreted pro-EmpA into mature EmpA. To identify the protease responsible for processing pro-EmpA, a minitransposon mutagenesis (using mini-Tn10Km) clone bank of V. anguillarum was screened for reduced protease activity due to insertions in undescribed genes. One mutant with reduced protease activity was identified. The region containing the mini-Tn10Km was cloned, sequenced, and found to contain epp, an open reading frame encoding a putative protease. Further characterization of epp was done using strain M101, created by single-crossover insertional mutagenesis. Protease activity was absent in M101 cultures even when empA protease activity was induced by salmon gastrointestinal mucus. When the epp mutation was complemented with a wild-type copy of epp (M102), protease activity was restored. Western blot analysis of sterile filtered culture supernatants from wild-type (M93Sm) cells, M101 cells, and M102 cells revealed that only pro-EmpA was present in M101supernatants; both pro-EmpA and mature EmpA were detected in M93Sm and M102 supernatants. When sterile filtered culture supernatants from the empA mutant strain (M99) and M101 were mixed, protease activity was restored. Western blot analysis revealed that pro-EmpA in M101 culture supernatant was processed to mature EmpA only after mixing with M99 culture supernatant. These data show that Epp is the EmpA-processing protease.
Varina
Maureen
M
Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA.
Denkin
Steven M
SM
Staroscik
Andrew M
AM
Nelson
David R
DR
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D023061:Gene Order; D005816:Genetic Complementation Test; D045726:Metalloproteases / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017422:Sequence Analysis, DNA; D014733:Vibrio / Q000201:enzymology* / Q000502:physiology*
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; C500552:EmpA protein, Vibrio anguillarum; D045726:Metalloproteases; D010447:Peptide Hydrolases