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 sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction.
Highlights
Pseudogymnoascus destructans is the fungus responsible for white nose syndrome (WNS) in bats [1,2,3]
More diffuse and slower hyphal growth was observed on defined minimal nutrient broth (MNB), requiring ~6 weeks to form a mycelial mat across the surface
The location of protease activity at the top of the gel MNB media indicated the protein migrated into the gel poorly, whereas the dominant activity in the tryptic soy broth (TSB) migrated midway down the gel, indicating distinct differences in size and charge for the associated protein
Summary
Pseudogymnoascus (basionym: Geomyces) destructans is the fungus responsible for white nose syndrome (WNS) in bats [1,2,3]. A clinical sign in WNS-infected bats is necrosis of the wing membrane, which may lead to infarction and electrolyte imbalances [6,7,8,9,10,11,12]. One hypothesis is that as bats exhibit frequent arousal from torpor, they “scratch” their wings to remove irritating fungal hyphae and physically damage tissues. Another hypothesis is P. destructans secretes proteases during infection, allowing mycelial penetration into underlying tissues. In addition to these possible roles in wing necrosis, immune reconstitution inflammatory syndrome (IRIS) in post-hibernating bats may exacerbate damage intensity [15]
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