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The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates.

Kennan RM, Wong W, Dhungyel OP, Han X, Wong D, Parker D, Rosado CJ, Law RH, McGowan S, Reeve SB, Levina V, Powers GA, Pike RN, Bottomley SP, Smith AI, Marsh I, Whittington RJ, Whisstock JC, Porter CJ, Rood JI - PLoS Pathog. (2010)

Bottom Line: These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions.The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important.Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism.

View Article: PubMed Central - PubMed

Affiliation: Monash University, Clayton, Victoria, Australia.

ABSTRACT
Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by other bacterial pathogens of both humans and animals.

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Protease activity of D. nodosus wild-type and protease mutants.Total protease activity of 40 h culture supernatants was measured using azocasein as the substrate. The protease activity is expressed relative to the wild-type activity. Means and standard error of the mean (s.e.m.) are shown. (A) Total protease activity of the protease mutants and their complemented derivatives. Each of the mutants had significantly reduced (P<0.05, n = 3, t-test) protease activity compared to the wild-type strain VCS1703A, while the complemented strains, except for the bprV complemented strain, were not significantly different to wild-type. WT: Wild-type VCS1703A, Benign: the benign isolate CS101. (B) Total protease activity of double and triple protease mutants. WT: Wild-type VCS1703A, Triple: the aprV2 aprV5 bprV triple mutant. The designation (1) and (2) represents independently derived mutants. Other genotypes are as indicated. The protease activity of each of these mutants was significantly different to wild-type (P<0.05, n = 3, t-test).
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ppat-1001210-g001: Protease activity of D. nodosus wild-type and protease mutants.Total protease activity of 40 h culture supernatants was measured using azocasein as the substrate. The protease activity is expressed relative to the wild-type activity. Means and standard error of the mean (s.e.m.) are shown. (A) Total protease activity of the protease mutants and their complemented derivatives. Each of the mutants had significantly reduced (P<0.05, n = 3, t-test) protease activity compared to the wild-type strain VCS1703A, while the complemented strains, except for the bprV complemented strain, were not significantly different to wild-type. WT: Wild-type VCS1703A, Benign: the benign isolate CS101. (B) Total protease activity of double and triple protease mutants. WT: Wild-type VCS1703A, Triple: the aprV2 aprV5 bprV triple mutant. The designation (1) and (2) represents independently derived mutants. Other genotypes are as indicated. The protease activity of each of these mutants was significantly different to wild-type (P<0.05, n = 3, t-test).

Mentions: To assess the contribution of each of the three extracellular proteases to the overall protease activity of the virulent D. nodosus isolate VCS1703A, separate chromosomal mutants of each protease gene were constructed by allelic exchange events that involved double crossovers. To confirm that the observed phenotypes resulted from these mutations, the mutants were complemented by insertion of the wild-type protease genes into the chromosome. Quantitative protease assays of culture supernatants, using azocasein as the substrate, showed that mutation of the aprV5 and aprV2 genes reduced total protease activity by 71% and 39%, respectively (Figure 1A). Complementation with the respective wild-type genes returned total protease activity to wild-type levels; however, it was also observed that the complemented aprV5 strain tended to lose protease activity upon repeated subculture. Since only a 12% reduction (P<0.05) was observed in the bprV mutant, BprV does not appear to make a major contribution to total protease activity. These results indicate that AprV5, either directly or indirectly, makes the major contribution to total extracellular protease activity.


The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates.

Kennan RM, Wong W, Dhungyel OP, Han X, Wong D, Parker D, Rosado CJ, Law RH, McGowan S, Reeve SB, Levina V, Powers GA, Pike RN, Bottomley SP, Smith AI, Marsh I, Whittington RJ, Whisstock JC, Porter CJ, Rood JI - PLoS Pathog. (2010)

Protease activity of D. nodosus wild-type and protease mutants.Total protease activity of 40 h culture supernatants was measured using azocasein as the substrate. The protease activity is expressed relative to the wild-type activity. Means and standard error of the mean (s.e.m.) are shown. (A) Total protease activity of the protease mutants and their complemented derivatives. Each of the mutants had significantly reduced (P<0.05, n = 3, t-test) protease activity compared to the wild-type strain VCS1703A, while the complemented strains, except for the bprV complemented strain, were not significantly different to wild-type. WT: Wild-type VCS1703A, Benign: the benign isolate CS101. (B) Total protease activity of double and triple protease mutants. WT: Wild-type VCS1703A, Triple: the aprV2 aprV5 bprV triple mutant. The designation (1) and (2) represents independently derived mutants. Other genotypes are as indicated. The protease activity of each of these mutants was significantly different to wild-type (P<0.05, n = 3, t-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991261&req=5

ppat-1001210-g001: Protease activity of D. nodosus wild-type and protease mutants.Total protease activity of 40 h culture supernatants was measured using azocasein as the substrate. The protease activity is expressed relative to the wild-type activity. Means and standard error of the mean (s.e.m.) are shown. (A) Total protease activity of the protease mutants and their complemented derivatives. Each of the mutants had significantly reduced (P<0.05, n = 3, t-test) protease activity compared to the wild-type strain VCS1703A, while the complemented strains, except for the bprV complemented strain, were not significantly different to wild-type. WT: Wild-type VCS1703A, Benign: the benign isolate CS101. (B) Total protease activity of double and triple protease mutants. WT: Wild-type VCS1703A, Triple: the aprV2 aprV5 bprV triple mutant. The designation (1) and (2) represents independently derived mutants. Other genotypes are as indicated. The protease activity of each of these mutants was significantly different to wild-type (P<0.05, n = 3, t-test).
Mentions: To assess the contribution of each of the three extracellular proteases to the overall protease activity of the virulent D. nodosus isolate VCS1703A, separate chromosomal mutants of each protease gene were constructed by allelic exchange events that involved double crossovers. To confirm that the observed phenotypes resulted from these mutations, the mutants were complemented by insertion of the wild-type protease genes into the chromosome. Quantitative protease assays of culture supernatants, using azocasein as the substrate, showed that mutation of the aprV5 and aprV2 genes reduced total protease activity by 71% and 39%, respectively (Figure 1A). Complementation with the respective wild-type genes returned total protease activity to wild-type levels; however, it was also observed that the complemented aprV5 strain tended to lose protease activity upon repeated subculture. Since only a 12% reduction (P<0.05) was observed in the bprV mutant, BprV does not appear to make a major contribution to total protease activity. These results indicate that AprV5, either directly or indirectly, makes the major contribution to total extracellular protease activity.

Bottom Line: These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions.The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important.Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism.

View Article: PubMed Central - PubMed

Affiliation: Monash University, Clayton, Victoria, Australia.

ABSTRACT
Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by other bacterial pathogens of both humans and animals.

Show MeSH
Related in: MedlinePlus