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.

Figure 1. Zymogram analysis of extracellular protease activity.

Gelatin was used as a substrate overlay of supernatants separated by native polyacrylamide gel electrophoresis to screen AprV5 and AprV2 activity in 25 h TAS broth cultures from the wild-type strain VCS1703A (WT), the aprV5 mutant JIR3756 (aprV5), the aprV2 mutant JIR3743 (aprV2), the bprV mutant JIR3928 (bprV) and the complemented derivatives JIR3883 (aprV5(aprV5⁺)), JIR3900 (aprV2(aprV2⁺)) and JIR3930 (bprV(bprV⁺)), respectively.

Figure 2. Schematic representation of the C-terminal deleted AprV5 derivatives.

The number of amino acid residues in each derivative is indicated as are the pre-, pro-, mature and C-terminal extension (CTE) regions.

Figure 3. Quantitative analysis of protease activity of deletion mutants.

Total protease activity in culture supernatants was determined with azocasein as the substrate. The culture supernatants from 16 h, 25 h and 40 h TAS broth cultures of the wild type strain VCS1703A (WT), the aprV5 mutant JIR3756 (aprV5), the aprV5Δ478–595 strain JIR3947 (V5ΔC1), the aprV5Δ503–595 strain JIR3956 (V5ΔC2), the aprV5Δ560–595 strain JIR3969 (V5ΔC3) were analysed as well as their complemented derivatives: JIR3968 (V5ΔC1(V5⁺)), JIR3965 (V5ΔC2(V5⁺)) and JIR3978 (V5ΔC3(V5⁺)). All values were obtained from three independent biological samples. Error bars represent SEM.

Figure 4. Extracellular protease zymograms of the aprV5 C-terminal deletion strains.

Cultures from 16 h, 25 h and 40 h TAS broth cultures of the wild-type strain VCS1703A (WT), the aprV5Δ478–595 strain JIR3947 (aprV5ΔC1) and its complemented derivative JIR3968 (aprV5ΔC1(V5⁺)), the aprV5Δ503–595 strain JIR3956 (aprV5ΔC2) and its complemented derivative JIR3965 (aprV5ΔC2(V5⁺)) and the aprV5 mutant JIR3756 (aprV5) were examined. The profile of the aprV5Δ560–595 strain JIR3969 (aprV5ΔC3) is not shown but was identical to that of aprV5ΔC2.

Figure 5. Western immunoblotting of wild-type and mutants with specific protease antisera.

Concentrated culture supernatants (3 µg) from 16 h, 25 h and 40 h (from left to right ) TAS growth of the wild type strain VCS1703A (WT), the aprV5Δ478–595 strain JIR3947 (V5ΔC1), the aprV5Δ503–595 strain JIR3956 (V5ΔC2), the aprV5Δ560–595 strain JIR3969 (V5ΔC3) and their complemented derivatives of JIR3968 (V5ΔC1(V5⁺), JIR3965 (V5ΔC2(V5⁺) and JIR3978 (V5ΔC3(V5⁺) were separated on 4–15% gradient SDS-PAGE and analysed by Western immunoblotting with (A) AprV5 (1∶4000)-, (B) AprV2 (1∶300)- and (C) BprV (1∶4000)- antisera. The aprV5 mutant JIR3756 (aprV5), the aprV2 mutant JIR3743 (aprV2) and the bprV mutant JIR3928 (bprV) were included as controls. The two groups of larger proteins in the aprV5 preparation are boxed.