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

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.

Cyclodepsipeptide Biosynthesis in Hypocreales Fungi and Sequence Divergence of The Non-Ribosomal Peptide Synthase Genes

“…The phylogeny reconstructions of housekeeping sequences (ITS— Figure 2 , tef -1α— Figure 3 ) point to the ancestral biosynthesis of beauvericin independently diverging into biosynthesis of other compounds (enniatins, beauvenniatins, allobeauvericins) across different taxa. The evolutionary relationships suggested by beauvericin gene fragments seem to support the notion of B. felina as an early diverging beauvericin producer, however, the obtained sequence fragment closely matches the cyclosporin synthase recently elucidated by Xu et al [ 55 ], so verification by cloning, functional genomics and/or whole genome sequencing is likely needed to fully verify the synthase sequence. The divergent positioning of three Fusarium isolates (1337, 41.9.3, P36) as well as an unusual GenBank reference sequence for F. oxysporum f.sp.…”

Identification of the Antifungal Metabolite Chaetoglobosin P From Discosia rubi Using a Cryptococcus neoformans Inhibition Assay: Insights Into Mode of Action and Biosynthesis

“…Natural products extracts from fungi were systematically tested for temperature selective activity in a two-plate agar to identify agents with enhanced of growth inhibition at ≥37°C, an effect that could potentially mitigate in vivo virulence of C. neoformans ( Xu et al, 2018 ). Temperature-dependent C. neoformans bioactivity was observed from the extracts of TTI-0863 and TTI-0885 when grown in wheat and MMK2 (also in medium supermalt for TTI-0885) where bioactivity was enhanced at 37°C compared to the same assay at 25°C ( Supplementary Figure S3 ).…”

“…The assay for temperature-selective growth inhibition of C. neoformans was described previously ( Xu et al, 2018 ). Overnight cultures of Staphylococcus aureus ATCC 43300 grown in Luria-Bertani (LB) broth (Sigma-Aldrich), and C. albicans ATCC 10231 and C. neoformans H99 grown in YM broth (1% malt extract, 0.2% yeast extract in deionized H 2 O) at 37°C were diluted with ATCC 43300 sterile H 2 O to an OD 600 of 0.4 for S. aureus and 0.8 for C. albicans and C. neoformans .…”

“…Furthermore, the few antifungal agents currently in clinical development have not been chosen based on effectiveness against Cryptococcus ( Perfect, 2017 ; Van Daele et al, 2019 ). We hypothesize that a Cryptococcus -centric screening approach can result in identification of metabolites not generally detected in the well-worn Candida albicans -centric antifungal discovery paradigm ( Xu et al, 2018 ). Therefore, new agents displaying anti-cryptococcal activity with minimal mammalian toxicity would be high priority objectives.…”

Sphaerostilbellins, New Antimicrobial Aminolipopeptide Peptaibiotics from Sphaerostilbella toxica

“…As basidiomycetes, Cryptococcus species potentially offer unique biological perspectives to antifungal drug development compared to more broadly investigated ascomycete pathogens such as Candida and Aspergillus species. We have continued to screen metabolite-enriched fermentation extracts from unstudied ascomycete fungi and have tested these extracts in a growth-inhibition assay against the clinical strain C. neoformans H99 with the objective of identifying metabolites that inhibit its growth in vitro [ 6 , 7 ].…”