Aneuploidy and Acquired Antifungal Drug Resistance in Candida species

University Of Minnesota, Minneapolis MN

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Abstract

ABSTRACT Antifungal drug resistant Candida species pose an urgent antimicrobial threat worldwide. Patients with invasive Candida infections experience high mortality >40%, despite the use of modern antifungal therapies. Only three drug classes are frequently used to treat Candida infections: azoles, echinocandins, and polyenes. Resistance to all three classes is increasing. The acquisition of antifungal drug resistance is frequently associated with large genomic copy number changes in diverse fungal pathogens, including Candida species. We identified novel large accordion-like DNA amplifications in C. albicans that rapidly expand during adaptation to azoles in vitro, amplifying hundreds of genes to more than 10 copies. We also reported that 50% of azole resistant C. albicans clinical isolates are aneuploid, and that Candida auris aneuploids can evolve during on-patient antifungal therapy. Aneuploidy is also associated with antifungal tolerance, a poorly understood phenotype that enables Candida to grow slowly at high concentrations of all three drug classes, well above the minimum inhibitory concentration. We propose that aneuploidy-mediated antifungal tolerance is promoting the evolution of antifungal resistance. Despite the frequency of aneuploidy, the rate and dynamics of chromosome copy number changes during adaptation to antifungal drug are not known for any fungal pathogen. We developed several high throughput single-cell and population-level approaches to comprehensively quantify the emergence and spread of all genomic changes during the acquisition of antifungal resistance, in real time. Our multidisciplinary approach uniquely positions us to identify new mechanisms of acquired antifungal tolerance and resistance in a rigorous and reproducible way. For example, we identified the sterol biosynthesis pathway as a hotspot for acquired multidrug resistance and tolerance across diverse Candida species, creating a paradigm shift: Multidrug resistance can evolve during adaptation to a single antifungal drug class, due to acquisition of a single point mutation. The aims of this proposal take a new approach to identifying the mechanisms driving antifungal drug resistance: We will use a new single-cell flow cytometry assay to quantify the rate and dynamics of aneuploid events during adaptation to each drug class and determine the effect of drug class on the evolution of drug resistance (Aim 1). We will comprehensively identify genes that when amplified alter drug susceptibility in vitro and in vivo using a novel CRISPR-amplification approach (Aim 2). Finally, we will use comprehensive molecular approaches to determine the degree to which mutations in the sterol biosynthesis pathway cause multidrug resistance in diverse Candida species (Aim 3). This comparative analysis of acquired resistance will provide a strong foundation for work in other Candida species. Together the outcomes from these experiments will identify the mechanisms that underlie how Candida species acquire drug tolerance and resistance and can pave the way for developing therapeutics to reduce the significant mortality caused by diverse antifungal drug resistant Candida species.