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The structural and biochemical basis for host tissue destruction mediated by subtilisin-like proteases from the ovine footrot pathogen, dichelobacter nodosus.
thesisposted on 2017-02-14, 00:29 authored by Wong, Wilson
The objective of this study was to investigate the role of D. nodosus extracellular serine proteases in the pathogenesis of ovine footrot. Ovine footrot causes enormous economic lost worldwide. The disease varies in severity and is classified into virulent and benign forms. Virulent footrot is characterised by the separation of the horn from soft tissue, resulting in loss of body condition, and reduction in wool and meat production. In contrast, benign footrot results in only a mild inflammation of the interdigital skin and does not severely impact on the body condition of the infected sheep. The causative agent of ovine footrot is the Gram-negative, anaerobic bacterium, Dichelobacter nodosus (D. nodosus). It is classified into virulent and benign strains according to the severity of the disease they cause. Both virulent and benign strains secrete several subtilisin-like proteases (subtilases) that play essential roles in the pathogenesis of footrot through their ability to mediate tissue damage. Virulent strains secrete the subtilases AprV2, AprV5 and BprV, which contain minor amino acid variations to related proteases AprB2, AprB5 and BprB produced by the benign strains. The molecular basis of how these proteases mediate footrot pathogenesis has not been established. In this study, proteomic analysis using hoof isolated from a healthy sheep identified keratin as the major substrate degraded by D. nodosus subtilases, providing direct evidence that these secreted proteases mediate the destruction of the hoof tissue, leading to the characteristics of virulent footrot. To relate these data at the molecular level, the crystal structures and biochemical activities of AprV2 and BprV from virulent strains, AprB2 and BprB from benign strains were characterised. The data revealed that D. nodosus subtilases employ an unusual mechanism of substrate proteolysis, whereby a novel disulfide tethered exosite loop (I2 loop) is required for the efficient recognition and degradation of extracellular matrix proteins. Importantly, bioinformatics data revealed the possible presence of the I2 loop in subtilases from other medically important pathogens, suggesting the I2-loop exosite represents a previously hitherto overlooked mechanism for substrate recognition and proteolysis in the subtilisin-like protease family. In addition, crystal structure and biochemical analyses of the basic proteases (BprV and BprB) revealed that differences in the properties of the S1 pocket of the proteases were the underlying cause for their differential activities against components of the extracellular matrix. Whilst both proteases showed similar primary substrate specificity, apparent differences in kinetics of substrate turnover were observed between the two enzymes. BprV was more efficient in cleaving after Leu containing peptides than BprB, which combined with the structural data, defines the molecular basis for the higher proteolytic activity of BprV against components of the ECM. In conclusion, subtilases from virulent strains of D. nodosus use a combination of effective S1 pocket and I2 exosite-mediated substrate recognition, to effectively destroy the host extracellular matrix, leading to severe tissue destruction. The structural and primary substrate specificity data presented here will be valuable for the design of veterinary therapeutic agents to specifically target D.nodosus subtilases for the treatment of footrot infection.