title: Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome.
abstract: Pseudogymnoascus destructans is the causative agent of white-nose syndrome, a disease that has caused the deaths of millions of bats in North America. This psychrophilic fungus proliferates at low temperatures and targets hibernating bats, resulting in their premature arousal from stupor with catastrophic consequences. Despite the impact of white-nose syndrome, little is known about the fungus itself or how it infects its mammalian host. P. destructans is not amenable to genetic manipulation, and therefore understanding the proteins involved in infection requires alternative approaches. Here, we identify hydrolytic enzymes secreted by P. destructans, and use a novel and unbiased substrate profiling technique to define active peptidases. These experiments revealed that endopeptidases are the major proteolytic activities secreted by P. destructans, and that collagen, the major structural protein in mammals, is actively degraded by the secretome. A serine endopeptidase, hereby-named Destructin-1, was subsequently identified, and a recombinant form overexpressed and purified. Biochemical analysis of Destructin-1 showed that it mediated collagen degradation, and a potent inhibitor of peptidase activity was identified. Treatment of P. destructans-conditioned media with this antagonist blocked collagen degradation and facilitated the detection of additional secreted proteolytic activities, including aminopeptidases and carboxypeptidases. These results provide molecular insights into the secretome of P. destructans, and identify serine endopeptidases that have the clear potential to facilitate tissue invasion and pathogenesis in the mammalian host.
authors.affiliation: Department of Pharmaceutical Chemistry.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: O'Donoghue
authors.affiliation: Department of Pharmaceutical Chemistry.
authors.forename: Giselle M
authors.initials: GM
authors.lastname: Knudsen
authors.affiliation: Department of Microbiology and Immunology, Brown University, Providence, RI 02912.
authors.forename: Chapman
authors.initials: C
authors.lastname: Beekman
authors.affiliation: Department of Microbiology and Immunology, Brown University, Providence, RI 02912.
authors.forename: Jenna A
authors.initials: JA
authors.lastname: Perry
authors.affiliation: Department of Biochemistry and Biophysics, Department of Microbiology and Immunology, and ajohnson@cgl.ucsf.edu Richard_Bennett@brown.edu.
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Johnson
authors.affiliation: Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94012; and.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: DeRisi
authors.affiliation: Department of Pharmaceutical Chemistry.
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.affiliation: Department of Microbiology and Immunology, Brown University, Providence, RI 02912 ajohnson@cgl.ucsf.edu Richard_Bennett@brown.edu.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Bennett
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity*; D001483:Base Sequence; D020134:Catalytic Domain; D002685:Chiroptera / Q000382:microbiology*; D017364:Collagenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009181:Mycoses / Q000382:microbiology / Q000662:veterinary*; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014774:Virulence
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; D017364:Collagenases
keywords: connective tissue; fungi; pathogenesis; peptidase; secretome