Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans

PloS One
Evan L PannkukBrett J Savary

Abstract

White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the s...Continue Reading

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Citations

May 7, 2015·Proceedings of the National Academy of Sciences of the United States of America·Anthony J O'DonoghueRichard J Bennett
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Aug 21, 2021·Journal of Industrial Microbiology & Biotechnology·Victoria L KornJoan W Bennett

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Datasets Mentioned

BETA
ELR04656.1
ELR05098.1
ELR04060
ELR07576.1
ELR03877.1
ELR10046.1

Methods Mentioned

BETA
electrophoresis
PCR

Software Mentioned

SignalP
BLASTp
ProteinLynx Global Server
FirstGlance
Jmol
Phylogeny
Mascot
SCAN
. fr
Clustal Omega

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