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Appl Microbiol Biotechnol. 2018 Mar;102(5):2337-2350. doi: 10.1007/s00253-018-8792-0. Epub 2018 Feb 2.

Identification of cyclosporin C from Amphichorda felina using a Cryptococcus neoformans differential temperature sensitivity assay.

Author information

1
Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 1881 East Road, 3SCR6.4676, Houston, TX, 77054, USA.
2
College of Agricultural Resources and Environment, Heilongjiang University, Harbin, 150080, China.
3
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
4
LifeMine Therapeutics, 430 E. 29th Street, Suite 830, New York, NY, 10016, USA.
5
Department of Chemistry, University of Iowa, Iowa City, IA, 52242, USA.
6
Departments of Biochemistry and Medicine, Duke University Medical Center, Durham, NC, 27710, USA.
7
Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 1881 East Road, 3SCR6.4676, Houston, TX, 77054, USA. billsge@vt.edu.

Abstract

We used a temperature differential assay with the opportunistic fungal pathogen Cryptococcus neoformans as a simple screening platform to detect small molecules with antifungal activity in natural product extracts. By screening of a collection extracts from two different strains of the coprophilous fungus, Amphichorda felina, we detected strong, temperature-dependent antifungal activity using a two-plate agar zone of inhibition assay at 25 and 37 °C. Bioassay-guided fractionation of the crude extract followed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) identified cyclosporin C (CsC) as the main component of the crude extract responsible for growth inhibition of C. neoformans at 37 °C. The presence of CsC was confirmed by comparison with a commercial standard. We sequenced the genome of A. felina to identify and annotate the CsC biosynthetic gene cluster. The only previously characterized gene cluster for the biosynthesis of similar compounds is that of the related immunosuppressant drug cyclosporine A (CsA). The CsA and CsC gene clusters share a high degree of synteny and sequence similarity. Amino acid changes in the adenylation domain of the CsC nonribosomal peptide synthase's sixth module may be responsible for the substitution of L-threonine compared to L-α-aminobutyric acid in the CsA peptide core. This screening strategy promises to yield additional antifungal natural products with a focused spectrum of antimicrobial activity.

KEYWORDS:

Adenylation domain; Antifungal; Ascomycota; Coprophilous fungi; Genome; Hypocreales; Nonribosomal peptide synthetase; Secondary metabolites; Thermal adaption

PMID:
29396588
PMCID:
PMC5942556
DOI:
10.1007/s00253-018-8792-0
[Indexed for MEDLINE]
Free PMC Article

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