RNAi-dependent epimutation roles in antimicrobial drug resistance and pathogenesis

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Abstract. This proposal focuses on the human fungal pathogenic Mucor species complex, a group of related pathogens that cause devastating infections that are difficult to treat, with limited drug treatment options, and requiring surgical debridement in some patients. Over the past decade, we and others have advanced genomics, genetics, and animal models for this understudied group of microbial pathogens. We discovered the protein phosphatase calcineurin controls the dimorphic transition from yeast to hyphae required for Mucor pathogenesis, and through studies of FK506-resistant isolates discovered a novel mechanism of antimicrobial drug resistance. In previously published and preliminary studies, significant advances were achieved through our discovery of a novel mechanism of antifungal drug resistance called epimutation, whereby the RNAi pathway is activated and silences drug target genes. This pathway confers transient, unstable drug resistance, and resistant isolates rapidly revert to drug sensitivity in the absence of drug. Through genetic and molecular studies, we defined RNAi components required for epimutation, those that are dispensable for epimutation, and a novel category that inhibits formation of epimutations. The discovery of antimicrobial drug resistance mediated via epimutations has been generalized: 1) showing epimutation occurs in two different pathogenic Mucor species, 2) defining an alternative RNAi pathway controlling epimutation frequency and stability, 3) identifying epimutations in additional genes causing resistance to antifungal agents, and 4) documenting that epimutations persist during animal infection or arise after animal passage. These insights set the stage for studies proposed here to further define mechanisms of epimutation, and elucidate the impact of epimutations on microbial pathogen interactions with the host. In the current proposal, we hypothesize epimutation is a general process that operates across many eukaryotic microbial pathogens, and acts as a major force in antimicrobial drug resistance that controls target genes involved in drug action, genome stability, and pathogenesis of eukaryotic microbial pathogens. Our studies will reveal unique facets of RNAi that lead to epimutations, which mediate antimicrobial drug resistance in ubiquitous fungal pathogens of humans. Aim 1 will 1) elucidate molecular mechanisms of epimutation and targets, including genes involved in drug resistance (including clinically used antifungal drugs) and transposable elements, and their impact on genome stability, 2) define conditions, including stress, sexual reproduction, and infection, that may drive the emergence of epimutations, and 3) establish the generalizability of these findings to other pathogenic fungal species. Aim 2 will define the impact of epimutation on antimicrobial drug resistance and pathogenicity in microbe interactions with immune cells, the blood-brain barrier, organoids, and whole-animal models. These studies will advance our understanding of how antimicrobial drug resistance can evolve via a novel RNAi-based pathway with direct implications for infectious disease evolution, treatment, and prevention, and provide insights into other eukaryotic pathogens with active RNAi pathways.