title: Antifungal drug resistance evoked via RNAi-dependent epimutations.
abstract: 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 calcineurin. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth. 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.
authors: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors: Silvia
authors: S
authors: Calo
authors: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors: Cecelia
authors: C
authors: Shertz-Wall
authors: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors: Soo Chan
authors: SC
authors: Lee
authors: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors: Robert J
authors: RJ
authors: Bastidas
authors: 1] Regional Campus of International Excellence "Campus Mare Nostrum", Murcia 30100, Spain [2] Department of Genetics and Microbiology, Faculty of Biology, University of Murcia, Murcia 30100, Spain.
authors: Francisco E
authors: FE
authors: Nicolás
authors: 1] Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA [2] Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North Carolina 27710, USA [3] Duke Center for the Genomics of Microbial Systems, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors: Joshua A
authors: JA
authors: Granek
authors: High-Throughput Sequencing Facility, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors: Piotr
authors: P
authors: Mieczkowski
authors: Department of Genetics and Microbiology, Faculty of Biology, University of Murcia, Murcia 30100, Spain.
authors: Santiago
authors: S
authors: Torres-Martínez
authors: Department of Genetics and Microbiology, Faculty of Biology, University of Murcia, Murcia 30100, Spain.
authors: Rosa M
authors: RM
authors: Ruiz-Vázquez
authors: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors: Maria E
authors: ME
authors: Cardenas
authors: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors: Joseph
authors: J
authors: Heitman
mesh_terms: D019703:Calcineurin / Q000235:genetics / Q000378:metabolism; D065095:Calcineurin Inhibitors; D025141:Drug Resistance, Fungal / Q000235:genetics*; D044127:Epigenesis, Genetic / Q000235:genetics*; D006801:Humans; D025301:Hyphae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D009089:Mucor / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D009091:Mucormycosis / Q000188:drug therapy / Q000382:microbiology; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D034622:RNA Interference; D016559:Tacrolimus / Q000378:metabolism / Q000494:pharmacology*; D022061:Tacrolimus Binding Protein 1A / Q000172:deficiency / Q000235:genetics / Q000378:metabolism
chemical_list: D065095:Calcineurin Inhibitors; D019703:Calcineurin; D022061:Tacrolimus Binding Protein 1A; D016559:Tacrolimus