Abstract
Pseudogymnoascus destructans is a pathogenic fungus responsible for White-nose Syndrome (WNS), a disease afflicting multiple species of North American bats. Pseudogymnoascus destructans infects susceptible bats during hibernation, invading dermal tissue and causing extensive tissue damage. In contrast, other Pseudogymnoascus species are non-pathogenic and cross-species comparisons may therefore reveal factors that contribute to virulence. In this study, we compared the secretome of P. destructans with that from several closely related Pseudogymnoascus species. A diverse set of hydrolytic enzymes were identified, including a putative serine peptidase, PdCP1, that was unique to the P. destructans secretome. A recombinant form of PdCP1 was purified and substrate preference determined using a multiplexed-substrate profiling method based on enzymatic degradation of a synthetic peptide library and analysis by mass spectrometry. Most peptide substrates were sequentially truncated from the carboxyl-terminus revealing that this enzyme is a bona fide carboxypeptidase. Peptides with arginine located close to the carboxyl-terminus were rapidly cleaved, and a fluorescent substrate containing arginine was therefore used to characterize PdCP1 activity and to screen a selection of peptidase inhibitors. Antipain and leupeptin were found to be the most potent inhibitors of PdCP1 activity.
Funding source: National Science Foundation
Award Identifier / Grant number: NSF-1456787
Funding statement: We thank Matt Ravalin for his advice on inhibitors of PdCP1. Funding for this project was provided by a National Science Foundation, Funder ID (10.13039/100000001), grant (NSF-1456787) to R.J.B. and Skaggs School of Pharmacy and Pharmacutical Sciences to A.J.O. Funding for J.M.P. and D.L.L. was provided by the Northern Research Station, USDA Forest Service. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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