Microorganisms evolve via a range of mechanisms that may include or involve sexual/parasexual reproduction, mutators, aneuploidy, Hsp90 and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show that the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidylprolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin1. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth2. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the fkbA gene, giving rise to drug-resistant epimutants. FK506-resistant epimutants readily reverted to the drug-sensitive wild-type phenotype when grown without exposure to the drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNAs and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves the generation of a double-stranded RNA trigger intermediate using the fkbA mature mRNA as a template to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes.
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Gene Expression Omnibus
Sequences for the fkbA gene from WT strain NRRL3631 and epimutant strains (EM1, EM2 and EM3) were deposited in GenBank with accession numbers KF203228, KF203229, KF203230 and KF203231. Raw data from high-throughput sRNA sequencing of WT, epimutant and revertant strains have been deposited in NCBI’s Gene Expression Omnibus and are accessible through accession number GSE56353.
We thank R. Skalsky and V. Ponnusamy for technical support and J. Wöstemeyer for trisporic acid. We thank B. Cullen, T. Petes, B. Billmyre, M. Feretzaki, J. Kingsbury and V. Ponnusamy for critical reading. This work was supported by NIH grants R37 AI39115-17, R01 AI50438-10, R01 CA154499-04 and the Spanish MICINN BFU2009-07220 and MINECO BFU2012-32246, co-financed by FEDER.
Extended data figures
The Supplementary Information contains 8 Tables that were too long to be included as Extended Data. Each table contains a small legend to explain the content included. These tables include the following information: Tables 1, 3 and 5: lists of the mutations found in the fkbA and calcineurin genes sequenced in every strain/isolate used for this study Table 2: 5′ and 3′ RACE sequences addressed in the manuscript Table 4: frequency of epimutants/mutants after exposure to stress conditions (experiment carried out to answer comments from the reviewers) Table 6: primers used in this study Table 7: adapter sequences used for sRNA libraries Table 8: basic library data