Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred

12 Issues per year


IMPACT FACTOR 2017: 3.022

CiteScore 2017: 2.81

SCImago Journal Rank (SJR) 2017: 1.562
Source Normalized Impact per Paper (SNIP) 2017: 0.705

Online
ISSN
1437-4315
See all formats and pricing
More options …
Volume 399, Issue 12

Issues

Characterization of PdCP1, a serine carboxypeptidase from Pseudogymnoascus destructans, the causal agent of White-nose Syndrome

Chapman Beekman
  • Department of Molecular Microbiology and Immunology, Brown University, 171 Meeting Street, Providence, RI 02912, USA
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Zhenze Jiang
  • Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Brian M. Suzuki
  • Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Jonathan M. Palmer
  • Center for Forest Mycology Research, Northern Research Station, USDA Forest Service, Madison, WI, USA
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Daniel L. Lindner
  • Center for Forest Mycology Research, Northern Research Station, USDA Forest Service, Madison, WI, USA
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Anthony J. O’Donoghue
  • Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Giselle M. Knudsen
  • Alaunus Biosciences, Inc., San Francisco, CA, USA
  • Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Richard J. BennettORCID iD: http://orcid.org/0000-0002-3277-9827
Published Online: 2018-10-26 | DOI: https://doi.org/10.1515/hsz-2018-0240

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.

This article offers supplementary material which is provided at the end of the article.

Keywords: bat infection; peptidase; proteomics; secretome; virulence

References

  • Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 8.Google Scholar

  • Baldo, A., Tabart, J., Vermout, S., Mathy, A., Collard, A., Losson, B., and Mignon, B. (2008). Secreted subtilisins of Microsporum canis are involved in adherence of arthroconidia to feline corneocytes. J. Med. Microbiol. 57, 1152–1156.Google Scholar

  • Baldo, A., Monod, M., Mathy, A., Cambier, L., Bagut, E.T., Defaweux, V., Symoens, F., Antoine, N., and Mignon, B. (2012). Mechanisms of skin adherence and invasion by dermatophytes. Mycoses 55, 218–223.Google Scholar

  • Behnsen, J., Lessing, F., Schindler, S., Wartenberg, D., Jacobsen, I.D., Thoen, M., Zipfel, P.F., and Brakhage, A.A. (2010). Secreted Aspergillus fumigatus protease Alp1 degrades human complement proteins C3, C4, and C5. Infect. Immun. 78, 3585–3594.Google Scholar

  • Blank, I.H. (1939). Measurement of pH of the skin surface. J. Invest. Dermatol. 2, 231–234.Google Scholar

  • Boyles, J.G., Cryan, P.M., Mccracken, G.F., Kunz, T.H. (2011). Economic importance of bats in agriculture. Science 332, 2.Google Scholar

  • Breddam, K. (1986). Serine carboxypeptidases: a review. Carlsberg Res. Commun. 51, 45.Google Scholar

  • Campbell, L.T., Simonin, A.R., Chen, C., Ferdous, J., Padula, M.P., Harry, E., Hofer, M., Campbell, I.L., and Carter, D.A. (2015). Cryptococcus strains with different pathogenic potentials have diverse protein secretomes. Eukaryot. Cell 14, 554–563.Google Scholar

  • Casadevall, A., Steenbergen, J.N., and Nosanchuk, J.D. (2003). ‘Ready made’ virulence and ‘dual use’ virulence factors in pathogenic environmental fungi – the Cryptococcus neoformans paradigm. Curr. Opin. Microbiol. 6, 332–337.Google Scholar

  • Chaturvedi, V., Springer, D.J., Behr, M.J., Ramani, R., Li, X., Peck, M.K., Ren, P., Bopp, D.J., Wood, B., Samsonoff, W.A., et al. (2010). Morphological and molecular characterizations of psychrophilic fungus Geomyces destructans from New York bats with White Nose Syndrome (WNS). PLoS One 5, e10783.Google Scholar

  • Colaert, N., Helsens, K., Martens, L., Vandekerckhove, J., and Gevaert, K. (2009). Improved visualization of protein consensus sequences by iceLogo. Nat. Methods 6, 786–787.Google Scholar

  • Cotrin, S.S., Puzer, L., De Souza Judice, W.A., Juliano, L., Carmona, A.K., and Juliano, M.A. (2004). Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides to define substrate specificity of carboxydipeptidases; assays with human cathepsin B. Anal. Biochem. 335, 244–252.Google Scholar

  • Elias, J.E., and Gygi, S.P. (2007). Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214.Google Scholar

  • Guan, S., Price, J.C., Prusiner, S.B., Ghaemmaghami, S., and Burlingame, A.L. (2011). A data processing pipeline for mammalian proteome dynamics studies using stable isotope metabolic labeling. Mol. Cell Proteomics 10, M111.010728.Google Scholar

  • Jousson, O., Lechenne, B., Bontems, O., Mignon, B., Reichard, U., Barblan, J., Quadroni, M., and Monod, M. (2004). Secreted subtilisin gene family in Trichophyton rubrum. Gene 339, 79–88.Google Scholar

  • Kaminishi, H., Miyaguchi, H., Tamaki, T., Suenaga, N., Hisamatsu, M., Mihashi, I., Matsumoto, H., Maeda, H., and Hagihara, Y. (1995). Degradation of humoral host defense by Candida albicans proteinase. Infect. Immun. 63, 984–988.Google Scholar

  • Koskinen, P., Toronen, P., Nokso-Koivisto, J., and Holm, L. (2015). PANNZER; high-throughput functional annotation of uncharacterized proteins in an error-prone environment. Bioinformatics 31, 1544–1552.Google Scholar

  • Krappmann, S. (2016). How to invade a susceptible host: cellular aspects of aspergillosis. Curr. Opin. Microbiol. 34, 136–146.Google Scholar

  • Krijger, J.J., Thon, M.R., Deising, H.B., and Wirsel, S.G. (2014). Compositions of fungal secretomes indicate a greater impact of phylogenetic history than lifestyle adaptation. BMC Genomics 15, 722.Google Scholar

  • Kunugi, S., Fukuda, M., and Hayashi, R. (1985). Action of serine carboxypeptidases on endopeptidase substrates, peptide-4-methyl-coumaryl-7-amides. Eur. J. Biochem. 153, 4.Google Scholar

  • Leopardi, S., Blake, D., and Puechmaille, S.J. (2015). White-nose syndrome fungus introduced from Europe to North America. Curr. Biol. 25, R217–R219.Google Scholar

  • Lowe, R.G.T. and Howlett, B.J. (2012). Indifferent, affectionate, or deceitful: lifestyles and secretomes of fungi. PLoS Pathog. 8, e1002515.Google Scholar

  • Maine, J.J. and Boyles, J.G. (2015). Bats initiate vital agroecological interactions in corn. Proc. Natl. Acad. Sci. USA 112, 12438–12443.Google Scholar

  • Martinez-Rossi, N.M., Peres, N.T., and Rossi, A. (2017). Pathogenesis of dermatophytosis: sensing the host tissue. Mycopathologia 182, 215–227.Google Scholar

  • Matousek, J.L. and Campbell, K.L. (2002). A comparative review of cutaneous pH. Vet. Dermatol. 13, 293–300.Google Scholar

  • Meteyer, C.U., Buckles, E.L., Blehert, D.S., Hicks, A.C., Green, D.E., Shearn-Bochsler, V., Thomas, N.J., Gargas, A., and Behr, M.J. (2009). Histopathologic criteria to confirm white-nose syndrome in bats. J. Vet. Diagn. Invest. 21, 411–414.Google Scholar

  • Mikesh, L.M., Aramadhaka, L.R., Moskaluk, C., Zigrino, P., Mauch, C., and Fox, J.W. (2013). Proteomic anatomy of human skin. J. Proteomics 84, 190–200.Google Scholar

  • Monod, M. (2008). Secreted proteases from dermatophytes. Mycopathologia 166; 285–294.Google Scholar

  • Monod, M., Capoccia, S., Lechenne, B., Zaugg, C., Holdom, M., and Jousson, O. (2002). Secreted proteases from pathogenic fungi. Int. J. Med. Microbiol. 292, 405–419.Google Scholar

  • Morschhauser, J., Virkola, R., Korhonen, T.K., and Hacker, J. (1997). Degradation of human subendothelial extracellular matrix by proteinase-secreting Candida albicans. FEMS Microbiol. Lett. 153, 349–355.Google Scholar

  • O’Donoghue, A.J., Eroy-Reveles, A.A., Knudsen, G.M., Ingram, J., Zhou, M., Statnekov, J.B., Greninger, A.L., Hostetter, D.R., Qu, G., Maltby, D.A., et al. (2012). Global identification of peptidase specificity by multiplex substrate profiling. Nat. Methods 9, 1095–1100.Google Scholar

  • O’Donoghue, A.J., Knudsen, G.M., Beekman, C., Perry, J.A., Johnson, A.D., Derisi, J.L., Craik, C.S., and Bennett, R.J. (2015). Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome. Proc. Natl. Acad. Sci. USA 112, E3152.Google Scholar

  • Ollert, M.W., Sohnchen, R., Korting, H.C., Ollert, U., Brautigam, S., and Brautigam, W. (1993). Mechanisms of adherence of Candida albicans to cultured human epidermal-keratinocytes. Infect. Immun. 61, 4560–4568.Google Scholar

  • Olsen, J.V., De Godoy, L.M., Li, G., Macek, B., Mortensen, P., Pesch, R., Makarov, A., Lange, O., Horning, S., and Mann, M. (2005). Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell. Proteomics 4, 2010–2021.Google Scholar

  • Palmer, J.M., Drees, K.P., Foster, J.T., and Lindner, D.L. (2018). Extreme sensitivity to ultraviolet light in the fungal pathogen causing white-nose syndrome of bats. Nat. Commun. 9, 35.Google Scholar

  • Pannkuk, E.L., Risch, T.S., and Savary, B.J. (2015). Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans. PLoS One 10, e0120508.Google Scholar

  • Pikula, J., Amelon, S.K., Bandouchova, H., Bartonicka, T., Berkova, H., Brichta, J., Hooper, S., Kokurewicz, T., Kolarik, M., Kollner, B., et al. (2017). White-nose syndrome pathology grading in Nearctic and Palearctic bats. PLoS One 12, e0180435.Google Scholar

  • Puechmaille, S.J., Wibbelt, G., Korn, V., Fuller, H., Forget, F., Muhldorfer, K., Kurth, A., Bogdanowicz, W., Borel, C., Bosch, T., et al. (2011). Pan-European distribution of white-nose syndrome fungus (Geomyces destructans) not associated with mass mortality. PLoS One 6, e19167.Google Scholar

  • Rawlings, N.D., Waller, M., Barrett, A.J., and Bateman, A. (2014). MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 42, D503–D509.Google Scholar

  • Reichard, U., Lechenne, B., Asif, A.R., Streit, F., Grouzmann, E., Jousson, O., and Monod, M. (2006). Sedolisins, a new class of secreted proteases from Aspergillus fumigatus with endoprotease or tripeptidyl-peptidase activity at acidic pHs. Appl. Environ. Microbiol. 72, 1739–1748.Google Scholar

  • Remington, S.R. (1993). Serine carboxypeptidases: a new and versatile family of enzymes. Curr. Opin. Biotechnol. 4, 7.Google Scholar

  • Reynolds, H.T. and Barton, H.A. (2014). Comparison of the white-nose syndrome agent Pseudogymnoascus destructans to cave-dwelling relatives suggests reduced saprotrophic enzyme activity. PLoS One 9, e86437.Google Scholar

  • Reynolds, H.T., Ingersoll, T., and Barton, H.A. (2015). Modeling the environmental growth of Pseudogymnoascus destructans and its impact on the white-nose syndrome epidemic. J. Wildl. Dis. 51, 318–331.Google Scholar

  • Sanglard, D., Hube, B., Monod, M., Odds, F.C., and Gow, N.A.R. (1997). A triple deletion of the secreted aspartyl proteinase genes SAP4, SAP5, and SAP6 of Candida albicans causes attenuated virulence. Infect. Immun. 65, 3539–3546.Google Scholar

  • Srinivasan, R.J.V.G., Angadiyavar, C.S. and Dreyfus, R.W. (1974). Anomalous fluorescence and laser emission from 7-alkylaminocoumarins in acid solutions. Chem. Phys. Lett. 25, 537–540.Google Scholar

  • Sriranganadane, D., Waridel, P., Salamin, K., Feuermann, M., Mignon, B., Staib, P., Neuhaus, J.M., Quadroni, M., and Monod, M. (2011). Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH. Proteomics 11, 4422–4433.Google Scholar

  • Takeuchi, M., Takagi, Y., Ebisui, R., Toyama, T., and Ichishima, E. (2014). Mode of action on fluorogenic substrates of acid carboxy-peptidases from Aspergillus. Agricult. Biol. Chem. 53, 1177–1178.Google Scholar

  • Thekkiniath, J.C., Zabet-Moghaddam, M., San Francisco, S.K., and San Francisco, M.J. (2013). A novel subtilisin-like serine protease of Batrachochytrium dendrobatidis is induced by thyroid hormone and degrades antimicrobial peptides. Fungal. Biol. 117, 451–461.Google Scholar

  • U.S. Fish and Wildlife Service. (2018). White-nose syndrome fact sheet: the devastating disease of hibernating bats in North America. Updated 7/2/2018.Google Scholar

  • Warnecke, L., Turner, J.M., Bollinger, T.K., Misra, V., Cryan, P.M., Blehert, D.S., Wibbelt, G., and Willis, C.K. (2013). Pathophysiology of white-nose syndrome in bats: a mechanistic model linking wing damage to mortality. Biol. Lett. 9, 20130177.Google Scholar

  • Wilson, M.B., Held, B.W., Freiborg, A.H., Blanchette, R.A., and Salomon, C.E. (2017). Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans, the cause of white-nose syndrome in bats. PLoS One 12, e0178968.Google Scholar

  • Yike, I. (2011). Fungal proteases and their pathophysiological effects. Mycopathologia 171, 299–323.Google Scholar

  • Zaugg, C., Jousson, O., Lechenne, B., Staib, P., and Monod, M. (2008). Trichophyton rubrum secreted and membrane- associated carboxypeptidases. Int. J. Med. Microbiol. 298, 669–682.Google Scholar

  • Zukal, J., Bandouchova, H., Brichta, J., Cmokova, A., Jaron, K.S., Kolarik, M., Kovacova, V., Kubatova, A., Novakova, A., Orlov, O., et al. (2016). White-nose syndrome without borders: Pseudogymnoascus destructans infection tolerated in Europe and Palearctic Asia but not in North America. Sci. Rep. 6, 19829.Google Scholar

About the article

Received: 2018-05-01

Accepted: 2018-09-24

Published Online: 2018-10-26

Published in Print: 2018-11-27


Funding Source: National Science Foundation

Award identifier / Grant number: NSF-1456787

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.


Citation Information: Biological Chemistry, Volume 399, Issue 12, Pages 1375–1388, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/hsz-2018-0240.

Export Citation

©2018 Walter de Gruyter GmbH, Berlin/Boston.Get Permission

Supplementary Article Materials

Comments (0)

Please log in or register to comment.
Log in