Applied Microbiology and Biotechnology

, Volume 102, Issue 5, pp 2337–2350 | Cite as

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

  • Lijian Xu
  • Yan Li
  • John B. Biggins
  • Brian R. Bowman
  • Gregory L. Verdine
  • James B. Gloer
  • J. Andrew Alspaugh
  • Gerald F. Bills
Applied microbial and cell physiology


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.


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



This work was supported by University of Texas Health Science Center at Houston new faculty start-up funds, the Kay and Ben Fortson Endowment (to G.B.), the Chinese Scholarship Council (to L.X.), and a grant from the NIH (R01 GM121458). Genome sequencing and assembly services were provided by New York Genome Center (New York, New York, USA) and A2IDEA (Ann Arbor, Michigan, USA), respectively.

Compliance with ethical standards

Conflict of interest

J.B.B., B.R.B., and G.L.V. have financial interests in Lifemine Therapeutics. None of the other authors declare any potential conflicts of interest.

Ethical approval

No work appearing in this article involved studies with human participants or animals.

Supplementary material

253_2018_8792_MOESM1_ESM.pdf (435 kb)
ESM 1 (PDF 434 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Texas Therapeutics Institute, The Brown Foundation Institute of Molecular MedicineThe University of Texas Health Science Center at HoustonHoustonUSA
  2. 2.College of Agricultural Resources and EnvironmentHeilongjiang UniversityHarbinChina
  3. 3.Institute of Vegetables and FlowersChinese Academy of Agricultural SciencesBeijingChina
  4. 4.LifeMine TherapeuticsNew YorkUSA
  5. 5.Department of ChemistryUniversity of IowaIowa CityUSA
  6. 6.Departments of Biochemistry and MedicineDuke University Medical CenterDurhamUSA

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