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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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Related in: MedlinePlus

Elastase activity of AprV2, AprB2 and mutants.For quantitative measurement of elastase activity of recombinant AprV2, AprB2 (AprV2.Y92R) and protease mutants, purified protease was incubated with Elastin-Congo Red at 25°C for 19 h. Elastin degradation was detected spectroscopically at 490 nm. The mean and s.e.m are shown (* p<0.001, n = 3, one-way ANOVA compared to AprV2).
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ppat-1001210-g002: Elastase activity of AprV2, AprB2 and mutants.For quantitative measurement of elastase activity of recombinant AprV2, AprB2 (AprV2.Y92R) and protease mutants, purified protease was incubated with Elastin-Congo Red at 25°C for 19 h. Elastin degradation was detected spectroscopically at 490 nm. The mean and s.e.m are shown (* p<0.001, n = 3, one-way ANOVA compared to AprV2).

Mentions: The apparent difference in elastase activity between AprV2 and AprB2 was assessed in vitro using an Elastin-Congo Red substrate and purified recombinant proteins. Under these conditions, the ability of AprB2 to degrade the substrate was significantly less than AprV2 (p<0.05, Figure 2). Both proteases displayed similar activity against the soluble chromogenic elastase substrate, N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (AAPVn), with kinetic parameters that were of the same order of magnitude (Table 1). These results suggest that the Y92R substitution does not contribute directly to catalysis at the active site of the enzyme.


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)

Elastase activity of AprV2, AprB2 and mutants.For quantitative measurement of elastase activity of recombinant AprV2, AprB2 (AprV2.Y92R) and protease mutants, purified protease was incubated with Elastin-Congo Red at 25°C for 19 h. Elastin degradation was detected spectroscopically at 490 nm. The mean and s.e.m are shown (* p<0.001, n = 3, one-way ANOVA compared to AprV2).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991261&req=5

ppat-1001210-g002: Elastase activity of AprV2, AprB2 and mutants.For quantitative measurement of elastase activity of recombinant AprV2, AprB2 (AprV2.Y92R) and protease mutants, purified protease was incubated with Elastin-Congo Red at 25°C for 19 h. Elastin degradation was detected spectroscopically at 490 nm. The mean and s.e.m are shown (* p<0.001, n = 3, one-way ANOVA compared to AprV2).
Mentions: The apparent difference in elastase activity between AprV2 and AprB2 was assessed in vitro using an Elastin-Congo Red substrate and purified recombinant proteins. Under these conditions, the ability of AprB2 to degrade the substrate was significantly less than AprV2 (p<0.05, Figure 2). Both proteases displayed similar activity against the soluble chromogenic elastase substrate, N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (AAPVn), with kinetic parameters that were of the same order of magnitude (Table 1). These results suggest that the Y92R substitution does not contribute directly to catalysis at the active site of the enzyme.

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