Antifungal Drug Concentration Impacts the Spectrum of Adaptive Mutations in Candida albicans

Abstract

Invasive fungal infections are a leading global cause of human mortality. Only three major classes of antifungal drugs are widely used, and resistance to all three classes can arise rapidly. The most widely prescribed antifungal drug, fluconazole, disseminates rapidly and reaches a wide range of concentrations throughout the body. The impact of drug concentration on the spectrum and effect of mutations acquired during adaptation is not known for any fungal pathogen, and how the specific level of a given stress influences the distribution of beneficial mutations has been poorly explored in general. We evolved 144 lineages from three genetically distinct clinical isolates of Candida albicans to four concentrations of fluconazole (0, 1, 8, and 64 μg/ml) and performed comprehensive phenotypic and genomic comparisons of ancestral and evolved populations. Adaptation to different fluconazole concentrations resulted in distinct adaptive trajectories. In general, lineages evolved to drug concentrations close to their MIC50 (the level of drug that reduces growth by 50% in the ancestor) tended to rapidly evolve an increased MIC50 and acquired distinct segmental aneuploidies and copy number variations. By contrast, lineages evolved to drug concentrations above their ancestral MIC50 tended to acquire a different suite of mutational changes and increased in drug tolerance (the ability of a subpopulation of cells to grow slowly above their MIC50). This is the first evidence that different concentrations of drug can select for different genotypic and phenotypic outcomes in vitro and may explain observed in vivo drug response variation.

Step-wise evolution of azole resistance through copy number variation followed byKSR1loss of heterozygosity inCandida albicans

“…We recently identified a lineage (AMS4058) in an evolution experiment in which the drug sensitive progenitor strain SC5314 acquired FLC resistance after 10 passages in 1 µg/mL FLC, resulting in an increase in MIC50 from 0.5 µg/mL to >256 µg/mL FLC [47]. The AMS4058 lineage also had reduced filamentation in vitro and decreased virulence in a mouse model of disseminated candidiasis (S2 Fig A ,B).…”

“…The lineage described in Fig 1 was evolved as previously described in [47]. As detailed there, a single colony isolated from susceptible lab strain SC5314 was suspended in 1 mL sterile YPAD media and grown overnight at 30°C.…”

“…CNV is another important class of mutations frequently found in C. albicans strains that evolve under stress. Both whole-chromosome and segmental aneuploidy are associated with increased resistance and/or tolerance to antifungal drugs in clinical isolates and in laboratory evolution experiments [46][47][48]. In some cases, the mechanisms causing increased resistance via CNV have been identified.…”

“…For example, amplification of the left arm of chromosome (Chr) 5 occurs via an isochromosome structure (i(5L)) that provides two additional copies of the fluconazole (FLC) target ERG11 and of a transcriptional activator of drug efflux pumps, TAC1 ; the extra copies of these two genes largely explains the drug resistance in strains carrying the i(5L) karyotype [12,49]. While the specific mechanisms of resistance have not been elucidated for most CNVs, the recurrent association of increased resistance or tolerance with specific CNVs suggests that specific genes within these amplified regions are responsible for increased resistance [46,47,50,51]. As with LOH, identifying the mechanisms of drug resistance is complicated by the large number (often hundreds) of genes amplified within a given CNV, any of which could potentially contribute to resistance via different mechanisms.…”

Impact of inter-species hybridisation on antifungal drug response in theSaccharomycesgenus

“…The strong selection pressures of exposure to such agents can result in the propagation of antifungal resistance (Fisher et al 2018), the emergence of fluconazole resistant C. albicans strains is well documented in long-term fluconazole treatment in HIV positive individuals (Johnson et al 1995). Indeed, exposure to different concentrations of drugs can also influence the speed and nature of the adaptive response of pathogenic fungi and yeasts to antifungal drugs (Todd et al 2023). Therefore, to perform thorough investigations of the networks and pathways associated with antifungal resistance it is essential to carry out genome-wide investigations to sample the breadth of the cellular response to antifungal agents.…”