Mucormycosis—an emergent, deadly fungal infection—is difficult to treat, in part because the causative species demonstrate broad clinical antifungal resistance. However, the mechanisms underlying drug resistance in these infections remain poorly understood. Our previous work demonstrated that one major agent of mucormycosis, Mucor circinelloides , can develop resistance to the antifungal agents FK506 and rapamycin through a novel, transient RNA interference-dependent mechanism known as epimutation. Epimutations silence the drug target gene and are selected by drug exposure; the target gene is re-expressed and sensitivity is restored following passage without drug. This silencing process involves generation of small RNA (sRNA) against the target gene via core RNAi pathway proteins. To further elucidate the role of epimutation in the broad antifungal resistance of Mucor , epimutants were isolated that confer resistance to another antifungal agent, 5-fluoroorotic acid (5-FOA). We identified epimutant strains that exhibit resistance to 5-FOA without mutations in PyrF or PyrG, enzymes which convert 5-FOA into the active toxic form. Using sRNA hybridization as well as sRNA library analysis, we demonstrate that these epimutants harbor sRNA against either pyrF or pyrG , and further show that this sRNA is lost after reversion to drug sensitivity. We conclude that epimutation is a mechanism capable of targeting multiple genes, enabling Mucor to develop resistance to a variety of antifungal agents. Elucidation of the role of RNAi in epimutation affords a fuller understanding of mucormycosis. Furthermore, it improves our understanding of fungal pathogenesis and adaptation to stresses, including the evolution of drug resistance. Author summary The emerging infection mucormycosis causes high mortality in part because the major causative fungi, including Mucor circinelloides , are resistant to most clinically available antifungal drugs. We previously discovered an RNA interference-based resistance mechanism, epimutation, through which M . circinelloides develops transient resistance to the antifungal agent FK506 by altering endogenous RNA expression. We further characterize this novel mechanism by isolating epimutations in two genes that confer resistance to another antifungal agent, 5-fluoroorotic acid. Thus, we demonstrate epimutation can induce resistance to multiple antifungals by targeting a variety of genes. These results reveal epimutation plays a broad role enabling rapid and reversible fungal responses to environmental stresses, including drug exposure, and controlling antifungal drug resistance and RNA expression. As resistance to antifungals emerges, a deeper understanding of the causative mechanisms is crucial for improving treatment.
【초록키워드】 Treatment, Evolution, Mutation, Pathogenesis, Infection, drugs, Proteins, drug, RNA, Rapamycin, sensitivity, response, Mucormycosis, RNA interference, drug target, small RNA, drug resistance, adaptation, novel, Enzymes, fungi, mechanism, fungal, RNAi, Analysis, strain, Mucor, Fungal infection, antifungal agents, resistance mechanism, target gene, antifungal agent, enzyme, high mortality, passage, treat, FK506, antifungal drug resistance, reversion, sRNA, RNA expression, RNAi pathway, Genes, IMPROVE, selected, develop, clinically, variety, demonstrated, induce, cause, restored, 【제목키워드】 pathogen, fungal,