On Mechanisms of Resistance in the Human Fungal Pathogen Mucor circinelloides

Abstract

Fungal pathogens are an emerging cause of disease globally, and mycoses are among the most difficult infections to treat. Antifungal drugs are limited in scope compared to antibiotics. Furthermore, many species of fungi display inherent or acquired antifungal resistance. Understanding the mechanisms by which these pathogens counter antifungals is crucial for both improving current treatment and the development of future antifungal agents. In this dissertation I have focused on the pathogen Mucor circinelloides, which is inherently resistant to almost the entire antifungal armamentarium. I have applied genetic techniques and sequencing to uncover several genetic and epigenetic factors which affect Mucor drug resistance in vitro, as well as established a new mouse model to study in vivo effects of resistance. In Chapter 1, I will briefly introduce fungal diseases and antifungal resistance, followed by a discussion of epigenetic factors which are known to confer to antifungal resistance in fungi. In Chapter 2, I will define the role played by a novel drug resistance pathway discovered in Mucor. The epimutation pathway is an RNA interference-dependent pathway, which enables Mucor to develop antifungal resistance by using its endogenous RNAi pathway to transiently suppress expression of drug target genes. Epimutation was previously discovered as a mechanism that could silence expression of one gene, fkbA, to confer resistance to the antifungal FK506. Here we demonstrated that epimutation is not limited to just one locus, but can confer drug resistance by targeting two other genetic loci, pyrF and pyrG; the PyrF or PyrG enzymes convert 5-FOA into the active toxic form, and silencing of either causes resistance to the antifungal 5-fluoroorotic acid (5-FOA). We identified epimutant strains that exhibit resistance to 5-FOA without mutations in either pyrF or pyrG. Using sRNA hybridization and 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.In Chapter 3, I developed a systemic, intravenous murine model of Mucor infection which we utilized to elucidate the role of epimutation in vivo. Infection with an epimutant strain resistant to the antifungal agents FK506 and rapamycin revealed that epimutant-induced drug resistance was stable in vivo in a variety of different organs and tissues. Reversion of the epimutant-induced drug resistance was observed to be more rapid in isolates from the brain, as compared to those recovered from the liver, spleen, kidney, or lungs. Importantly, infection with a wild-type strain of Mucor led to increased rates of epimutation after strains were recovered from organs and exposed to FK506 stress in vitro. Once again, this effect was more pronounced in strains recovered from the brain than from other organs. In Chapter 4, I describe a different mechanism through which Mucor can develop drug resistance – transposon movement. This is the first known report of transposon-induced resistance in Mucor. By studying the emergence of resistance to the antifungal agent FK506 in wild-type Mucor after passage through a murine model of infection, I identified a transposon that inserted in the promoter region of fkbA, the gene encoding the drug target of FK506. Sequencing of this insertion revealed a transposon that shares similarity with previously described Helitrons, a class of DNA transposons. In addition, we were able to sequence and identify a second putative transposon, a retrotransposon, which is also capable of disrupting the fkbA locus to induce FK506 resistance by inserting into the coding or promoter regions of this gene. This second case of transposon-induced drug resistance occurred across multiple independently-derived strains of RNA interference-deficient Mucor. In Chapter 5, I will conclude this thesis and discuss potential future directions which could extend from this work.