Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50D Mutant Susceptibility for Lead Compound Development

Robert J. Tancer, Yina Wang, Siddhi Pawar, Chaoyang Xue, Gregory R. Wiedman

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Cryptococcus neoformans is a major fungal pathogen that often causes life-threatening meningitis in immunocompromised populations. This yeast pathogen is highly resistant to the echinocandin drug caspofungin. Previous studies showed that Cryptococcus lipid translocase (flippase) is required for the caspofungin resistance of that fungus. Mutants with a deleted subunit of lipid flippase, Cdc50, showed increased sensitivity to caspofungin. Here we designed an antifungal peptide targeting the P4-ATPase function. We synthesized stable peptides based on the Cdc50 loop region to identify peptides that can sensitize caspofungin by blocking flippase function and found that myristylated peptides based on the "AS15 sequence" was effective at high concentrations. A modified peptide, "AW9-Ma" showed a MIC of 64 mg/mL against H99 wild type and a fractional inhibitory concentration (FIC) index value of 0.5 when used in combination with caspofungin. Most notably, in the presence of the AW9-Ma peptide, C. neoformans wild type was highly sensitive to caspofungin with a MIC of 4 mg/mL, the same as the cdc50D mutant. Further assays with flow cytometry showed inhibition of the lipid flippase enzyme activity and significant accumulation of phosphatidylserine on the cell membrane surface. Using a fluorescently labeled peptide, we confirmed that the peptide co-localized with mCherry-tagged P4- ATPase protein Apt1 in C. neoformans. Structure-activity relationship studies of the AW9 sequence showed that two lysine residues on the peptide are likely responsible for the interaction with the P4-ATPase, hence critical for its antifungal activity.

Original languageEnglish (US)
JournalMicrobiology Spectrum
Volume10
Issue number2
DOIs
StatePublished - Apr 2022

All Science Journal Classification (ASJC) codes

  • Physiology
  • Ecology
  • Immunology and Microbiology(all)
  • Genetics
  • Microbiology (medical)
  • Cell Biology
  • Infectious Diseases

Keywords

  • antimicrobial agents
  • antimicrobial peptides
  • Cryptococcus neoformans
  • drug synergy
  • fungal meningitis
  • fungi
  • immunocompromised hosts
  • lead compounds
  • mutants
  • resistance

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