Fungal Secretome Analysis via PepSAVI-MS: Identification of the Bioactive Peptide KP4 from Ustilago maydis

  • Christine L. Kirkpatrick
  • Nicole C. Parsley
  • Tessa E. Bartges
  • Madeline E. Cooke
  • Wilaysha S. Evans
  • Lilian R. Heil
  • Thomas J. Smith
  • Leslie M. Hicks
Focus: 29th Sanibel Conference, Peptidomics: Bridging the Gap Between Proteomics and Metabolomics by MS: Research Article


Fungal secondary metabolites represent a rich and largely untapped source for bioactive molecules, including peptides with substantial structural diversity and pharmacological potential. As methods proceed to take a deep dive into fungal genomes, complimentary methods to identify bioactive components are required to keep pace with the expanding fungal repertoire. We developed PepSAVI-MS to expedite the search for natural product bioactive peptides and herein demonstrate proof-of-principle applicability of the pipeline for the discovery of bioactive peptides from fungal secretomes via identification of the antifungal killer toxin KP4 from Ustilago maydis P4. This work opens the door to investigating microbial secretomes with a new lens, and could have broad applications across human health, agriculture, and food safety.

Graphical Abstract


Bioactive peptides Antimicrobial peptides (AMPs) Fungal secretomes Peptidomics Mass spectrometry Natural products 


  1. 1.
    Brundrett, M.C.: Coevolution of roots and mycorrhizas of land plants. New Phytol. 154, 275–304 (2002)CrossRefGoogle Scholar
  2. 2.
    Blackwell, M.: The fungi: 1, 2, 3 ... 5.1 million species? Am. J. Bot. 98, 426–438 (2011)CrossRefPubMedGoogle Scholar
  3. 3.
    Scherlach, K., Graupner, K., Hertweck, C.: Molecular bacteria-fungi interactions: effects on environment, food, and medicine. Annu. Rev. Microbiol. 67, 375–397 (2013)CrossRefPubMedGoogle Scholar
  4. 4.
    Dang, T., Süssmuth, R.D.: Bioactive peptide natural products as lead structures for medicinal use. Acc. Chem. Res. 50, 1566–1576 (2017)CrossRefPubMedGoogle Scholar
  5. 5.
    Mandal, S.M., Roy, A., Ghosh, A.K., Hazra, T.K., Basak, A., Franco, O.L.: Challenges and future prospects of antibiotic therapy: from peptides to phages utilization. Front. Pharmacol. 5, 105 (2014)CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Walensky, L.D., Bird, G.H.: Hydrocarbon-stapled peptides: principles, practice, and progress. J. Med. Chem. 57, 6275–6288 (2014)CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Essig, A., Hofmann, D., Munch, D., Gayathri, S., Kunzler, M., Kallio, P.T., Sahl, H.G., Wider, G., Schneider, T., Aebi, M.: Copsin, a novel peptide-based fungal antibiotic interfering with the peptidoglycan synthesis. J. Biol. Chem. 289, 34953–34964 (2014)CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Bomgardner, M.M.: Spider venom: An insecticide whose time has come? Chem. Eng. News. 95, 30–31 (2017)Google Scholar
  9. 9.
    Golneshin, A., Adetutu, E., Ball, A.S., May, B.K., Van, T.T., Smith, A.T.: Complete genome sequence of Lactobacillus plantarum strain B21, a bacteriocin-producing strain isolated from Vietnamese fermented sausage Nem Chua. Genome Announc. 3, e00055–e00015 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Clevenger, K.D., Bok, J.W., Ye, R., Miley, G.P., Verdan, M.H., Velk, T., Chen, C., Yang, K., Robey, M.T., Gao, P., Lamprecht, M., Thomas, P.M., Islam, M.N., Palmer, J.M., Wu, C.C., Keller, N.P., Kelleher, N.L.: A scalable platform to identify fungal secondary metabolites and their gene clusters. Nat. Chem. Biol. 13, 895–901 (2017)CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Grigoriev, I.V., Cullen, D., Goodwin, S.B., Hibbett, D., Jeffries, T.W., Kubicek, C.P., Kuske, C., Magnuson, J.K., Martin, F., Spatafora, J.W., Tsang, A., Baker, S.E.: Fueling the future with fungal genomics. Mycology. 2, 192–209 (2011)Google Scholar
  12. 12.
    Kirkpatrick, C.L., Broberg, C.A., McCool, E.N., Lee, W.J., Chao, A., McConnell, E.W., Pritchard, D.A., Hebert, M., Fleeman, R., Adams, J., Jamil, A., Madera, L., Stromstedt, A.A., Goransson, U., Liu, Y., Hoskin, D.W., Shaw, L.N., Hicks, L.M.: The “PepSAVI-MS” pipeline for natural product bioactive peptide discovery. Anal. Chem. 89, 1194–1201 (2017)CrossRefPubMedGoogle Scholar
  13. 13.
    Amaral, A.C., Silva, O.N., Mundim, N.C.C.R., de Carvalho, M.J.A., Migliolo, L., Leite, J.R.S.A., Prates, M.V., Bocca, A.L., Franco, O.L., Felipe, M.S.S.: Predicting antimicrobial peptides from eukaryotic genomes: In silico strategies to develop antibiotics. Peptides. 37, 301–308 (2012)CrossRefPubMedGoogle Scholar
  14. 14.
    Mygind, P.H., Fischer, R.L., Schnorr, K.M., Hansen, M.T., Sonksen, C.P., Ludvigsen, S., Raventos, D., Buskov, S., Christensen, B., De Maria, L., Taboureau, O., Yaver, D., Elvig-Jorgensen, S.G., Sorensen, M.V., Christensen, B.E., Kjaerulff, S., Frimodt-Moller, N., Lehrer, R.I., Zasloff, M., Kristensen, H.H.: Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Nature. 437, 975–980 (2005)CrossRefPubMedGoogle Scholar
  15. 15.
    Allen, A., Islamovic, E., Kaur, J., Gold, S., Shah, D., Smith, T.J.: Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut. Plant Biotechnol. J. 9, 857–864 (2011)CrossRefPubMedGoogle Scholar
  16. 16.
    Gage, M.J., Rane, S.G., Hockerman, G.H., Smith, T.J.: The virally encoded fungal toxin KP4 specifically blocks L-type voltage-gated calcium channels. Mol. Pharmacol. 61, 936–944 (2002)CrossRefPubMedGoogle Scholar
  17. 17.
    Vizcaino, J.A., Csordas, A., Del-Toro, N., Dianes, J.A., Griss, J., Lavidas, I., Mayer, G., Perez-Riverol, Y., Reisinger, F., Ternent, T., Xu, Q.W., Wang, R., Hermjakob, H.: 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44, D447–D456 (2016)CrossRefPubMedGoogle Scholar
  18. 18.
    Gu, F., Khimani, A., Rane, S.G., Flurkey, W.H., Bozarth, R.F., Smith, T.J.: Structure and function of a virally encoded fungal toxin from Ustilago maydis: a fungal and mammalian Ca2+ channel inhibitor. Structure. 3, 805–814 (1995)CrossRefPubMedGoogle Scholar
  19. 19.
    Gu, F., Khimani, A., Rane, S., Flurkey, W.H., Bozarth, R.F., Smith, T.J.: The structure of a virally encoded fungal toxin from Ustilago maydis that inhibits fungal and mammalian calcium channels. Springer, Berlin (1996)CrossRefGoogle Scholar
  20. 20.
    Gu, F., Sullivan, T.S., Che, Z., Ganesa, C., Flurkey, W.H., Bozarth, R.F., Smith, T.J.: The characterization and crystallization of a virally encoded Ustilago maydis KP4 toxin. J. Mol. Biol. 243, 792–795 (1994)CrossRefPubMedGoogle Scholar
  21. 21.
    Tao, J., Ginsberg, I., Banerjee, N., Held, W., Koltin, Y., Bruenn, J.A.: Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins. Mol. Cell. Biol. 10, 1373–1381 (1990)CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Gage, M.J., Bruenn, J., Fischer, M., Sanders, D., Smith, T.J.: KP4 fungal toxin inhibits growth in Ustilago maydis by blocking calcium uptake. Mol. Microbiol. 41, 775–785 (2001)CrossRefPubMedGoogle Scholar
  23. 23.
    Allen, A., Snyder, A.K., Preuss, M., Nielsen, E.E., Shah, D.M., Smith, T.J.: Plant defensins and virally encoded fungal toxin KP4 inhibit plant root growth. Planta. 227, 331–339 (2008)CrossRefPubMedGoogle Scholar
  24. 24.
    Mohimani, H., Kersten, R.D., Liu, W.T., Wang, M., Purvine, S.O., Wu, S., Brewer, H.M., Pasa-Tolic, L., Bandeira, N., Moore, B.S., Pevzner, P.A., Dorrestein, P.C.: Automated genome mining of ribosomal peptide natural products. ACS Chem. Biol. 9, 1545–1551 (2014)CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Medema, M.H., Paalvast, Y., Nguyen, D.D., Melnik, A., Dorrestein, P.C., Takano, E., Breitling, R.: Pep2Path: automated mass spectrometry-guided genome mining of peptidic natural products. PLoS Comput. Biol. 10, e1003822 (2014)CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  • Christine L. Kirkpatrick
    • 1
  • Nicole C. Parsley
    • 1
  • Tessa E. Bartges
    • 1
  • Madeline E. Cooke
    • 1
  • Wilaysha S. Evans
    • 1
  • Lilian R. Heil
    • 1
  • Thomas J. Smith
    • 2
  • Leslie M. Hicks
    • 1
  1. 1.Department of ChemistryThe University of North Carolina at Chapel HillChapel HillUSA
  2. 2.Department of Biochemistry and Molecular BiologyThe University of Texas Medical BranchGalvestonUSA

Personalised recommendations