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

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.

Fig 1. Pseudogymnoascus destructans growth in static liquid cultures.

Left panel depicts typical culture morphology in minimal nutrient broth with gelatin (A) compared to a nutritionally complex tryptic soy broth (B). The right panel depicts a SDS-PAGE (Coomassie-Brilliant Blue G-250 stain) of extracellular proteins recovered from culture media and a native gel zymogram (casein) illustrates protease activity from total protein extract.

Fig 2. Total protease activity (units x 10³/ml) secreted by P. destructans grown in static liquid cultures.

Various protein-supplemented minimal nutrient broth media and complex culture media were used. Controls are total activity accumulated in culture medium, and residual activity was determined following treatment with the protease inhibitors EDTA, PMSF, and E-64. Minimal nutrient broth with K, keratin, G, gelatin, E, elastin, C, casein. Complex media: TSB, tryptic soy broth, TP, tryptone peptone, BHI, brain-heart infusion, PP, proteose peptone. (Mean and standard error for triplicate samples.)

Fig 3. Preparative IEF separation and analysis of P. destructans extracellular proteins.

Secreted proteins were recovered from MBN-G culture medium with broad-range ampholytes. Activity profile separated with broad-range ampholytes in the Rotofor cell (A). pH gradient indicated by dashed line. Protease activity assayed with FITC-casein: activity indicates fluorescence units per ml. Pooled activity peak fractions (10–13) resolved by SDS-PAGE and stained with Coomassie-brilliant blue.

Fig 4. SDS-PAGE of P. destructans extracellular proteins separated by ConA lectin affinity chromatography.

Lane 1, Biorad Precision Plus mass standards; lane 2, Crude enzyme substrate; lane 3, ConA elution (from non-adjacent gel lane); lane 4, western blot analysis detection in enriched protein concentrate using a PdSP1 antiserum.

Fig 5. Peptide mass fingerprint and MS/MS spectra of PdSP1.

MALDI-TOF MS spectrum (m/z 700–3000) from PdSP1 tryptic peptides (A). MS/MS spectrum with b/y-series ions from peptide ion m/z 908.49 (Ox+16) (SVISMSLR) (B).

Fig 6. Sequence alignment of Pseudogymnoascus spp. Family S8 serine proteases.

Sequences identified from GenBank accessions: P. destructans, PdSP3, ELR10046.1; P. destructans, PdSP1, ELR07576.1; P. destructans, PdSP2, ELR03877.1; and P. pannorum KFZ06449.1. Location of catalytic triad, D₁₆₀, H₁₉₂, and S₃₄₅ (indicated by * below sequence) with conserved motifs for S8A subfamily indicated in gray boxes. Residues predicted to participate in calcium-binding are also indicated below sequences for sites C1 (+) and C2 (#). Amino acids sequences determined experimentally from PdSP1 are underlined. N-glycosylation sequons (N-X-S/T) are indicated in bold italics. Alignment prepared with ClustalW Omega.

Fig 7. Phylogenetic tree generated from S8 serine proteases primary sequence from P. destructans and other select microbial organisms.

Included are three serine proteases from P. destructans, selected serine protease sequences (S8A proteinase-K subfamily) from plant pathogenic and human dermatophytic fungi, proteinase K, Carlsberg subtilisin, and Aspergillus cavatus serine protease. Phylogenetic tree generated with program Phylogeny.fr (S8A accessions are listed in S1 Fig.)