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Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development

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...

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Autores principales: Tancer, Robert J., Wang, Yina, Pawar, Siddhi, Xue, Chaoyang, Wiedman, Gregory R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9045296/
https://www.ncbi.nlm.nih.gov/pubmed/35377230
http://dx.doi.org/10.1128/spectrum.00439-22
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author Tancer, Robert J.
Wang, Yina
Pawar, Siddhi
Xue, Chaoyang
Wiedman, Gregory R.
author_facet Tancer, Robert J.
Wang, Yina
Pawar, Siddhi
Xue, Chaoyang
Wiedman, Gregory R.
author_sort Tancer, Robert J.
collection PubMed
description 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 μg/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 μg/mL, the same as the cdc50Δ 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. IMPORTANCE The authors have developed a lead compound peptide antifungal drug targeting a protein from the organism Cryptococcus neoformans. Binding of the drug to the target fungal protein causes charged lipid molecules to be retained on the surface. This peptide works in synergy with the existing antifungal drug caspofungin. Echinocandin drugs like caspofungin are one of the few classes of existing antifungals. Due to the high concentrations needed, caspofungin is rarely used to treat C. neoformans infections. The authors believe that their new compound provides a way to lower the concentration of caspofungin needed to treat such infections, thus opening the possibility for greater utility of these antifungal.
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spelling pubmed-90452962022-04-28 Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development Tancer, Robert J. Wang, Yina Pawar, Siddhi Xue, Chaoyang Wiedman, Gregory R. Microbiol Spectr Research Article 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 μg/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 μg/mL, the same as the cdc50Δ 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. IMPORTANCE The authors have developed a lead compound peptide antifungal drug targeting a protein from the organism Cryptococcus neoformans. Binding of the drug to the target fungal protein causes charged lipid molecules to be retained on the surface. This peptide works in synergy with the existing antifungal drug caspofungin. Echinocandin drugs like caspofungin are one of the few classes of existing antifungals. Due to the high concentrations needed, caspofungin is rarely used to treat C. neoformans infections. The authors believe that their new compound provides a way to lower the concentration of caspofungin needed to treat such infections, thus opening the possibility for greater utility of these antifungal. American Society for Microbiology 2022-04-04 /pmc/articles/PMC9045296/ /pubmed/35377230 http://dx.doi.org/10.1128/spectrum.00439-22 Text en Copyright © 2022 Tancer et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Tancer, Robert J.
Wang, Yina
Pawar, Siddhi
Xue, Chaoyang
Wiedman, Gregory R.
Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development
title Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development
title_full Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development
title_fullStr Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development
title_full_unstemmed Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development
title_short Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development
title_sort development of antifungal peptides against cryptococcus neoformans; leveraging knowledge about the cdc50δ mutant susceptibility for lead compound development
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9045296/
https://www.ncbi.nlm.nih.gov/pubmed/35377230
http://dx.doi.org/10.1128/spectrum.00439-22
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