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Substrate specifity profiling of the Aspergillus fumigatus proteolytic secretome reveals consensus motifs with predominance of Ile/Leu and Phe/Tyr.

Watson DS, Feng X, Askew DS, Jambunathan K, Kodukula K, Galande AK - PLoS ONE (2011)

Bottom Line: A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints.However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus.

View Article: PubMed Central - PubMed

Affiliation: Center for Advanced Drug Research, Biosciences Division, SRI International, Harrisonburg, Virginia, United States of America.

ABSTRACT

Background: The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician's toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers.

Methodology and principal findings: As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.

Conclusions: This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis.

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Comparative activity of secreted proteases of Aspergillus strains and species.A. Cleavage of individual IQFPs by Aspergillus culture supernatants. The brightness of a given square corresponds to the extent of cleavage of the IQFP probe in that well, as described in Figure 2. Fluorescence measurements were normalized by the biomass of each culture according to dry weight to facilitate absolute comparisons across strains. Data represent the mean of two independent experiments. A. fumigatus exhibited the greatest extent of proteolytic activity, followed by A. nidulans, then A. flavus. IQFP probes used for the experiments in panels B and C are highlighted in red. B. Cleavage of selected IQFPs by three Aspergillus species. Values represent the mean of three strains per species. C. Effect of heating at 50°C for 30 min on the proteolytic activity of A. fumigatus H237 and A. nidulans A4 culture supernatants. A. nidulans supernatant retained >80% of activity under these conditions, whereas A. fumigatus supernatant retained only 30–40%. * p≤0.01 as compared to A. fumigatus. For panels A and B, data represent fluorescence fold change after 2 h incubation; for panel C, data represent fluorescence fold change after 1 h incubation. Error bars represent standard deviations.
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pone-0021001-g007: Comparative activity of secreted proteases of Aspergillus strains and species.A. Cleavage of individual IQFPs by Aspergillus culture supernatants. The brightness of a given square corresponds to the extent of cleavage of the IQFP probe in that well, as described in Figure 2. Fluorescence measurements were normalized by the biomass of each culture according to dry weight to facilitate absolute comparisons across strains. Data represent the mean of two independent experiments. A. fumigatus exhibited the greatest extent of proteolytic activity, followed by A. nidulans, then A. flavus. IQFP probes used for the experiments in panels B and C are highlighted in red. B. Cleavage of selected IQFPs by three Aspergillus species. Values represent the mean of three strains per species. C. Effect of heating at 50°C for 30 min on the proteolytic activity of A. fumigatus H237 and A. nidulans A4 culture supernatants. A. nidulans supernatant retained >80% of activity under these conditions, whereas A. fumigatus supernatant retained only 30–40%. * p≤0.01 as compared to A. fumigatus. For panels A and B, data represent fluorescence fold change after 2 h incubation; for panel C, data represent fluorescence fold change after 1 h incubation. Error bars represent standard deviations.

Mentions: In two independent experiments, we observed that protease substrate specificity patterns were consistent within species and no significant intraspecies variation was observed (Figure 7A). The lone exception was A. fumigatus strain CEA10, which had greater proteolytic activity than the other two A. fumigatus strains, although the cleavage patterns were consistent. In contrast, several important differences were observed when comparing species. Overall, proteolytic activity was generally greater in A. fumigatus strains, followed by A. nidulans, with A. flavus exhibiting the lowest level of proteolytic cleavage of the three species (Figure 7B). A. terreus proteolytic cleavage was undetectable at the dilution studied (data not shown). Differences in substrate specificities were also observed among species; cleavage preferences for the Ser/Thr-Ile/Leu-Asn/Gln and Ala/Val-Ile/Leu-Ile/Leu motifs were consistent, but interspecies differences in substrate specificities of the other two motifs were evident. While A. fumigatus proteases cleaved Ser/Thr-Ile/Leu-Phe/Tyr promiscuously, A. nidulans proteases preferred sequences containing Ile at the Yaa position and A. flavus proteases preferred sequences containing Leu at the Yaa position. In addition, cleavage of the Ile/Leu-Phe/Tyr-Phe/Tyr motif was detectable in culture supernatants of all three A. flavus isolates but almost entirely absent in A. nidulans culture supernatants.


Substrate specifity profiling of the Aspergillus fumigatus proteolytic secretome reveals consensus motifs with predominance of Ile/Leu and Phe/Tyr.

Watson DS, Feng X, Askew DS, Jambunathan K, Kodukula K, Galande AK - PLoS ONE (2011)

Comparative activity of secreted proteases of Aspergillus strains and species.A. Cleavage of individual IQFPs by Aspergillus culture supernatants. The brightness of a given square corresponds to the extent of cleavage of the IQFP probe in that well, as described in Figure 2. Fluorescence measurements were normalized by the biomass of each culture according to dry weight to facilitate absolute comparisons across strains. Data represent the mean of two independent experiments. A. fumigatus exhibited the greatest extent of proteolytic activity, followed by A. nidulans, then A. flavus. IQFP probes used for the experiments in panels B and C are highlighted in red. B. Cleavage of selected IQFPs by three Aspergillus species. Values represent the mean of three strains per species. C. Effect of heating at 50°C for 30 min on the proteolytic activity of A. fumigatus H237 and A. nidulans A4 culture supernatants. A. nidulans supernatant retained >80% of activity under these conditions, whereas A. fumigatus supernatant retained only 30–40%. * p≤0.01 as compared to A. fumigatus. For panels A and B, data represent fluorescence fold change after 2 h incubation; for panel C, data represent fluorescence fold change after 1 h incubation. Error bars represent standard deviations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3117871&req=5

pone-0021001-g007: Comparative activity of secreted proteases of Aspergillus strains and species.A. Cleavage of individual IQFPs by Aspergillus culture supernatants. The brightness of a given square corresponds to the extent of cleavage of the IQFP probe in that well, as described in Figure 2. Fluorescence measurements were normalized by the biomass of each culture according to dry weight to facilitate absolute comparisons across strains. Data represent the mean of two independent experiments. A. fumigatus exhibited the greatest extent of proteolytic activity, followed by A. nidulans, then A. flavus. IQFP probes used for the experiments in panels B and C are highlighted in red. B. Cleavage of selected IQFPs by three Aspergillus species. Values represent the mean of three strains per species. C. Effect of heating at 50°C for 30 min on the proteolytic activity of A. fumigatus H237 and A. nidulans A4 culture supernatants. A. nidulans supernatant retained >80% of activity under these conditions, whereas A. fumigatus supernatant retained only 30–40%. * p≤0.01 as compared to A. fumigatus. For panels A and B, data represent fluorescence fold change after 2 h incubation; for panel C, data represent fluorescence fold change after 1 h incubation. Error bars represent standard deviations.
Mentions: In two independent experiments, we observed that protease substrate specificity patterns were consistent within species and no significant intraspecies variation was observed (Figure 7A). The lone exception was A. fumigatus strain CEA10, which had greater proteolytic activity than the other two A. fumigatus strains, although the cleavage patterns were consistent. In contrast, several important differences were observed when comparing species. Overall, proteolytic activity was generally greater in A. fumigatus strains, followed by A. nidulans, with A. flavus exhibiting the lowest level of proteolytic cleavage of the three species (Figure 7B). A. terreus proteolytic cleavage was undetectable at the dilution studied (data not shown). Differences in substrate specificities were also observed among species; cleavage preferences for the Ser/Thr-Ile/Leu-Asn/Gln and Ala/Val-Ile/Leu-Ile/Leu motifs were consistent, but interspecies differences in substrate specificities of the other two motifs were evident. While A. fumigatus proteases cleaved Ser/Thr-Ile/Leu-Phe/Tyr promiscuously, A. nidulans proteases preferred sequences containing Ile at the Yaa position and A. flavus proteases preferred sequences containing Leu at the Yaa position. In addition, cleavage of the Ile/Leu-Phe/Tyr-Phe/Tyr motif was detectable in culture supernatants of all three A. flavus isolates but almost entirely absent in A. nidulans culture supernatants.

Bottom Line: A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints.However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus.

View Article: PubMed Central - PubMed

Affiliation: Center for Advanced Drug Research, Biosciences Division, SRI International, Harrisonburg, Virginia, United States of America.

ABSTRACT

Background: The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician's toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers.

Methodology and principal findings: As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.

Conclusions: This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis.

Show MeSH
Related in: MedlinePlus