Global analysis of genetic circuitry and adaptive mechanisms enabling resistance to the azole antifungal drugs

Abstract

Invasive fungal infections caused by the pathogen Candida albicans have transitioned from a rare curiosity to a major cause of human mortality. This is in part due to the emergence of resistance to the limited number of antifungals available to treat fungal infections. Azoles function by targeting the biosynthesis of ergosterol, a key component of the fungal cell membrane. Loss-of-function mutations in the ergosterol biosynthetic gene ERG3 mitigate azole toxicity and enable resistance that depends upon fungal stress responses. Here, we performed a genome-wide synthetic genetic array screen in Saccharomyces cerevisiae to map ERG3 genetic interactors and uncover novel circuitry important for azole resistance. We identified nine genes that enabled erg3-mediated azole resistance in the model yeast and found that only two of these genes had a conserved impact on resistance in C. albicans. Further, we screened a C. albicans homozygous deletion mutant library and identified 13 genes for which deletion enhances azole susceptibility. Two of the genes, RGD1 and PEP8, were also important for azole resistance acquired by diverse mechanisms. We discovered that loss of function of retrograde transport protein Pep8 overwhelms the functional capacity of the stress response regulator calcineurin, thereby abrogating azole resistance. To identify the mechanism through which the GTPase activator protein Rgd1 enables azole resistance, we selected for mutations that restore resistance in strains lacking Rgd1. Whole genome sequencing uncovered parallel adaptive mechanisms involving amplification of both chromosome 7 and a large segment of chromosome 3. Overexpression of a transporter gene on the right portion of chromosome 3, NPR2, was sufficient to enable azole resistance in the absence of Rgd1. Thus, we establish a novel mechanism of adaptation to drug-induced stress, define genetic circuitry underpinning azole resistance, and illustrate divergence in resistance circuitry over evolutionary time.

Identifying Candida albicans Gene Networks Involved in Pathogenicity

“…We find that pep8 cells produce delayed or stunted hyphae and show attenuated macrophage escape (Figures 5, 6), confirming a role for this gene in filamentation as previously reported (Uhl et al, 2003) and suggesting a role in infection. This finding also confirms recent work, which is entirely independent from this study, that has shown that pep8 mutants demonstrate severe filamentation defects in a range of conditions (Azadmanesh et al, 2017) and these mutants abrogate azole resistance (Mount et al, 2018). Loss of function of Pep8 abrogates azole resistance by overwhelming the functional capacity of calcineurin.…”

Expandable and reversible copy number amplification drives rapid adaptation to antifungal drugs

“…To further address the mechanism of formation and to understand the impact of copy number on fitness, we used a set of isogenic isolates obtained from an agar plate instead of liquid cultures. These four single colony isolates (AMS3051, AMS3052, AMS3053, and AMS3054) were obtained from the same progenitor (AMS3050) after 120 hr growth on a single miconazole (20 μg/ml) agar plate ( Mount et al, 2018 ). Prior whole genome sequencing analysis of these colonies identified a shared CNV of Chr3L ( Mount et al, 2018 ).…”

“…These four single colony isolates (AMS3051, AMS3052, AMS3053, and AMS3054) were obtained from the same progenitor (AMS3050) after 120 hr growth on a single miconazole (20 μg/ml) agar plate ( Mount et al, 2018 ). Prior whole genome sequencing analysis of these colonies identified a shared CNV of Chr3L ( Mount et al, 2018 ). To test the hypothesis that these single colonies represented different outcomes from the same recombination event, due to the short exposure to miconazole and similarity in karyotypes, we first performed read depth analysis to characterize the CNV breakpoints.…”

Candida albicans Genetic Background Influences Mean and Heterogeneity of Drug Responses and Genome Stability during Evolution in Fluconazole

“…Growth in fluconazole is expected to exert selection pressure for some aneuploids more than others; specific genes within an aneuploid chromosome that are responsible for increased drug resistance (26) and drug tolerance (27) also have been identified. In many other cases, more than one gene may contribute to the phenotype (22,27,48,68,69). While we assume that changes in DNA content of a given isolate are largely due to chromosomal aneuploidy, it is important to note that increased levels of mitochondrial and/or ribosomal DNA can also contribute to differences in DNA content level detected by flow cytometry, as was recently shown to occur in different deletion mutants within the "isogenic" collection of Saccharomyces cerevisiae deletion mutants (70).…”