title: The AprV5 subtilase is required for the optimal processing of all three extracellular serine proteases from Dichelobacter nodosus.
abstract: Dichelobacter nodosus is the principal causative agent of ovine footrot and its extracellular proteases are major virulence factors. Virulent isolates of D. nodosus secrete three subtilisin-like serine proteases: AprV2, AprV5 and BprV. These enzymes are each synthesized as precursor molecules that include a signal (pre-) peptide, a pro-peptide and a C-terminal extension, which are processed to produce the mature active forms. The function of the C-terminal regions of these proteases and the mechanism of protease processing and secretion are unknown. AprV5 contributes to most of the protease activity secreted by D. nodosus. To understand the role of the C-terminal extension of AprV5, we constructed a series of C-terminal-deletion mutants in D. nodosus by allelic exchange. The proteases present in the resultant mutants and their complemented derivatives were examined by protease zymogram analysis, western blotting and mass spectrometry. The results showed that the C-terminal region of AprV5 is required for the normal expression of protease activity, deletion of this region led to a delay in the processing of these enzymes. D. nodosus is an unusual bacterium in that it produces three closely related extracellular serine proteases. We have now shown that one of these enzymes, AprV5, is responsible for its own maturation, and for the optimal cleavage of AprV2 and BprV, to their mature active forms. These studies have increased our understanding of how this important pathogen processes these virulence-associated extracellular proteases and secretes them into its external environment.
authors.affiliation: Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria, Australia.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Han
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Kennan
authors.forename: David L
authors.initials: DL
authors.lastname: Steer
authors.forename: A Ian
authors.initials: AI
authors.lastname: Smith
authors.forename: James C
authors.initials: JC
authors.lastname: Whisstock
authors.forename: Julian I
authors.initials: JI
authors.lastname: Rood
mesh_terms: D000818:Animals; D002417:Cattle; D020376:Dichelobacter nodosus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D005535:Foot Rot / Q000382:microbiology; D017353:Gene Deletion; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology / Q000662:veterinary; D011487:Protein Conformation; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D037521:Virulence Factors; D057057:Serine Proteases