Antifungal Tolerance and Resistance Emerge at Distinct Drug Concentrations and Rely upon Different Aneuploid Chromosomes

Yang, Feng; Costa, Eduardo Scopel Ferreira da; Li, Hao; Sun, Liu-Liu; Kawar, Nora; Cao, Yongbing; Jiang, Yuanying; Berman, Judith

doi:10.1128/mbio.00227-23

Cited by 21 publications

(22 citation statements)

References 47 publications

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“…These findings provide additional evidence that a distinct mechanism of FLU resistance distinguishes cluster 1 from clusters 2 and 3. The implication that different resistance mechanisms will dominate evolution in slightly different concentrations of the same drug highlights the complexity of adaptation and the need to more deeply understand the diversity of potential adaptive mechanisms before designing treatment strategies (Berman and Krysan 2020; Yang et al 2023).…”

Section: Resultsmentioning

confidence: 99%

Distinguishing mutants that resist drugs via different mechanisms by examining fitness tradeoffs

Schmidlin,

Apodaca,

Newell

et al. 2023

Preprint

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There is growing interest in designing multidrug therapies that leverage tradeoffs to combat drug resistance. Tradeoffs are common in evolution and occur when, for example, resistance to one drug results in sensitivity to another. Major questions remain about the extent to which tradeoffs are reliable, specifically, whether the mutants that provide resistance to a given drug all suffer similar tradeoffs. This question is difficult because the drug-resistant mutants observed in the clinic, and even those evolved in controlled laboratory settings, are often biased towards those that provide large fitness benefits. Thus, the mutations (and mechanisms) that provide drug resistance may be more diverse than current data suggests. Here, we perform evolution experiments utilizing lineage-tracking to capture a fuller spectrum of mutations that give yeast cells a fitness advantage in fluconazole, a common antifungal drug. We then quantify fitness tradeoffs for each of 774 evolved mutants across 12 environments, finding these mutants group into six classes with characteristically different tradeoffs. Their unique tradeoffs may imply that each group of mutants affects fitness through different molecular mechanisms. Some of the groupings we find are surprising. For example, we find some mutants that resist single drugs do not resist their combination, and some mutants to the same gene have different tradeoffs than others. These findings, on one hand, demonstrate the difficulty in relying on consistent or intuitive tradeoffs when designing multidrug treatments that thwart resistance. On the other hand, by demonstrating that hundreds of adaptive mutations can be reduced to a relatively smaller number of groups, our findings suggest that resistance evolves through a relatively small number of mechanisms such that multidrug strategies that hinder resistance may be possible. Finally, by grouping mutants that likely affect fitness through similar underlying mechanisms, our findings also guide efforts to map the phenotypic impacts of mutation and predict the impacts of some mutations from others.

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Section: Resultsmentioning

confidence: 99%

Distinguishing mutants that resist drugs via different mechanisms by examining fitness tradeoffs

Schmidlin,

Apodaca,

Newell

et al. 2023

Preprint

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“…Aneuploidy is associated with the rapid evolution of azole tolerance [74,75] (Zhou et al 2024, under review). We recently found that the Chr3 and Chr6 concurrent aneuploidies conferred multi-azole tolerance via elevated drug efflux pumps (Zhou et al 2024, under review).…”

Section: Discussionmentioning

confidence: 99%

Erg251 has complex and pleiotropic effects on azole susceptibility, filamentation, and stress response phenotypes

Zhou,

Hilk,

Solis

et al. 2024

Preprint

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Ergosterol is essential for fungal cell membrane integrity and growth, and numerous antifungal drugs target ergosterol. Inactivation or modification of ergosterol biosynthetic genes can lead to changes in antifungal drug susceptibility, filamentation and stress response. Here, we found that the ergosterol biosynthesis gene ERG251 is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds of Candida albicans. Heterozygous point mutations led to single allele dysfunction of ERG251 and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance in C. albicans. Importantly, single allele dysfunction of ERG251 in combination with recurrent chromosome aneuploidies resulted in bona fide azole resistance. Homozygous deletions of ERG251 caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Dysfunction of ERG251 resulted in transcriptional upregulation of the alternate sterol biosynthesis pathway and ZRT2, a Zinc transporter. Notably, we determined that overexpression of ZRT2 is sufficient to increase azole tolerance in C. albicans. Our combined transcriptional and phenotypic analyses revealed the pleiotropic effects of ERG251 on stress responses including cell wall, osmotic and oxidative stress. Interestingly, while loss of either allele of ERG251 resulted in similar antifungal drug responses, we observed functional divergence in filamentation regulation between the two alleles of ERG251 (ERG251-A and ERG251-B) with ERG251-A exhibiting a dominant role in the SC5314 genetic background. Finally, in a murine model of systemic infection, homozygous deletion of ERG251 resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study provides extensive genetic, transcriptional and phenotypic analysis for the effects of ERG251 on drug susceptibility, fitness, filamentation and stress responses.

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“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Zande,

Gautier,

Kawar

et al. 2024

Preprint

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Antimicrobial drug resistance poses a global health threat, requiring a deeper understanding of the evolutionary processes that lead to its emergence in pathogens. Complex evolutionary dynamics involve multiple mutations that can result in cooperative or competitive (clonal interference) effects.Candida albicans, a major fungal pathogen, displays high rates of copy number variation (CNV) and loss of heterozygosity (LOH). CNV and LOH events involve large numbers of genes and could synergize during evolutionary adaptation. Understanding the contributions of CNV and LOH to antifungal drug adaptation is challenging, especially in the context of whole-population genome sequencing. Here, we document the sequential evolution of fluconazole tolerance and then resistance in aC. albicansisolate involving an initial CNV on chromosome 4, followed by an LOH on chromosome R that involvesKSR1. Similar LOH events involvingKSR1,which encodes a reductase involved in sphingolipid biosynthesis, were also detected in independently evolved fluconazole resistant isolates. We dissect the specificKSR1codons that affect fluconazole resistance and tolerance. The combination of the chromosome 4 CNV andKSR1LOH results in a >500-fold increase in azole resistance, illustrating a compelling example of rapid, yet step-wise, interplay between CNV and LOH in drug resistance evolution.

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Antifungal Tolerance and Resistance Emerge at Distinct Drug Concentrations and Rely upon Different Aneuploid Chromosomes

Cited by 21 publications

References 47 publications

Distinguishing mutants that resist drugs via different mechanisms by examining fitness tradeoffs

Distinguishing mutants that resist drugs via different mechanisms by examining fitness tradeoffs

Erg251 has complex and pleiotropic effects on azole susceptibility, filamentation, and stress response phenotypes

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

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