Genetic and genomic architecture of the evolution of resistance to antifungal drug combinations

Abstract

The evolution of drug resistance in fungal pathogens compromises the efficacy of the limited number of antifungal drugs. Drug combinations have emerged as a powerful strategy to enhance antifungal efficacy and abrogate drug resistance, but the impact on the evolution of drug resistance remains largely unexplored. Targeting the molecular chaperone Hsp90 or its downstream effector, the protein phosphatase calcineurin, abrogates resistance to the most widely deployed antifungals, the azoles, which inhibit ergosterol biosynthesis. Here, we evolved experimental populations of the model yeast Saccharomyces cerevisiae and the leading human fungal pathogen Candida albicans with azole and an inhibitor of Hsp90, geldanamycin, or calcineurin, FK506. To recapitulate a clinical context where Hsp90 or calcineurin inhibitors could be utilized in combination with azoles to render resistant pathogens responsive to treatment, the evolution experiment was initiated with strains that are resistant to azoles in a manner that depends on Hsp90 and calcineurin. Of the 290 lineages initiated, most went extinct, yet 14 evolved resistance to the drug combination. Drug target mutations that conferred resistance to geldanamycin or FK506 were identified and validated in five evolved lineages. Whole-genome sequencing identified mutations in a gene encoding a transcriptional activator of drug efflux pumps, PDR1, and a gene encoding a transcriptional repressor of ergosterol biosynthesis genes, MOT3, that transformed azole resistance of two lineages from dependent on calcineurin to independent of this regulator. Resistance also arose by mutation that truncated the catalytic subunit of calcineurin, and by mutation in LCB1, encoding a sphingolipid biosynthetic enzyme. Genome analysis revealed extensive aneuploidy in four of the C. albicans lineages. Thus, we identify molecular determinants of the transition of azole resistance from calcineurin dependence to independence and establish multiple mechanisms by which resistance to drug combinations evolves, providing a foundation for predicting and preventing the evolution of drug resistance.

Figure 1. Design and outcome of the experimental evolution of resistance to drug combinations.

A) Experimental populations were initiated with S. cerevisiae and C. albicans strains resistant to azoles due to erg3 loss of function. This resistance mechanism is contingent on Hsp90 and calcineurin, such that inhibition of either of these cellular stress response regulators results in cell death (t₀). Propagation of these strains in the presence of azole and the Hsp90 inhibitor geldanamycin or azole and the calcineurin inhibitor FK506 at concentrations that exert selection pressure for resistance to the drug combination results in the evolution of resistance to geldanamycin or FK506 (t_1a) or the evolution of an azole resistance mechanism that is independent of Hsp90 or calcineurin (t_1b) among extant lineages. B) Single colony founders were used to initiate evolution experiments in 24- or 96-well plates containing control and treatment wells. Controls consisted of: no drug, azole alone, geldanamycin alone, or FK506 alone, where drug concentrations were not inhibitory. Treatment wells consisted of combinations of azole and geldanamycin or FK506, selected based on dose response matrices (see Figure S2). C) Experimental evolution of resistance to azole and geldanamycin or azole and FK506 yielded 14 resistant lineages out of 290 initiated. Ca = Candida albicans; Sc = Saccharomyces cerevisiae.

Figure 2. The populations evolved distinct resistance profiles.

Levels of resistance to azole and FK506 (A, B) or azole and geldanamycin (C–E) of evolved strains of S. cerevisiae (A, C, D) and C. albicans (B, E), relative to their ancestors. Resistance was measured with a constant concentration of azole and a gradient of geldanamycin or FK506 in YPD at 30°C for 2 days (B) or 3 days (A, C–E). Optical densities were averaged for duplicate measurements and normalized relative to drug-free controls (see colour bar). GdA = geldanamycin and FL = fluconazole.

Figure 3. Cross-resistance profiles provide a strategy to predict resistance mechanisms.

(A) Strains evolved in azole and FK506 were tested for cross-resistance to azole and the calcineurin inhibitor cyclosporin A as well as azole and the Hsp90 inhibitor geldanamycin. (B) Candidate resistance mechanisms based on specific cross-resistance profiles of strains evolved with azole and FK506. (C) Strains evolved in azole and geldanamycin were tested for cross-resistance to azole and the Hsp90 inhibitor radicicol as well as azole and the calcineurin inhibitor FK506. (D) Candidate resistance mechanisms based on specific cross-resistance profiles of strains evolved with azole and geldanamycin. GdA = geldanamycin; RAD = radicicol; and CsA = cyclosporin A.

Figure 4. Mutations in HSP90 confer resistance to azole and geldanamycin in two S. cerevisiae lineages and in one C. albicans lineage.

(A) Sc-G-12 (right panel) and Sc-G-14 (left panel) are both resistant to azole and geldanamycin and slightly cross-resistant to azole and radicicol, relative to their parental strains (above). (B) Resistance to azole and geldanamycin in Sc-G-14 is attributable to HSC82^I117N. Replacing the ancestral allele with the HSC82^I117N allele expressed on a plasmid increases resistance of the ancestral strain to the level observed in Sc-G-14, while replacing the HSC82^I117N allele with the ancestral allele on a plasmid abrogates resistance of Sc-G-14. (C) Deletion of HSC82 in Sc-G-12 or its parental strain phenocopies resistance of Sc-G-12, suggesting that HSC82^K385* confers resistance by loss of function of HSC82. (D) Ca-G-10 has increased resistance to azole and geldanamycin but no cross-resistance to azole and FK506 or azole and radicicol. (E) Resistance to azole and geldanamycin in Ca-G-10 is attributable to HSP90^D91Y. Replacing the native HSP90 allele in parental strain with HSP90^D91Y phenocopied resistance of Ca-G-10. Conversely, resistance of Ca-G-10 was abrogated when HSP90^D91Y was replaced with the ancestral HSP90 allele. Resistance assays were performed and analyzed is in Figure 2, after incubation at 30°C for 2 days (D) or 3 days (A–C, E). Assays were performed in YPD (A, C–E) or SD with amino acid supplements (B). GdA = geldanamycin; RAD = radicicol; and FL = fluconazole.

Figure 5. Mutations in FPR1 confer resistance to azole and FK506 in two S. cerevisiae lineages.

Sc-F-3 (A) and Sc-F-2 (B) evolved resistance to azole and FK506 but no cross-resistance to azole and either geldanamycin or cyclosporin A. (C) FPR1^V108F confers resistance in Sc-F-3. Replacing the ancestral FPR1 allele with FPR1^V108F expressed from a plasmid increases resistance of the ancestral strain to a similar level as that observed in Sc-F-3. Conversely, replacing the FPR1^V108F allele of Sc-F-3 with the ancestral FPR1 allele expressed on a plasmid abrogates resistance of Sc-F-3. (D) FPR1^dupG53-D61 confers resistance in Sc-F-2. Replacing the ancestral FPR1 allele with FPR1^dupG53-D61 expressed from a plasmid increases resistance of the ancestral strain, while replacing the FPR1^dupG53-D61 allele of Sc-F-2 with the ancestral FPR1 allele expressed on a plasmid abrogates resistance of Sc-F-2. (E) The resistance phenotype of Sc-F-3 remains dependent on calcineurin. Deletion of the regulatory subunit of calcineurin necessary for its function, CNB1, abolished resistance to azole and FK506. (F) The resistance phenotype of Sc-F-2 remains dependent on calcineurin. Resistance assays were performed and analyzed is in Figure 2, with incubation for 2 days at 30°C in YPD (A, B, D, and F) or SD with amino acid supplements (C and E). CsA = cyclosporin A; GdA = geldanamycin; FL = fluconazole; and M = miconazole.

Figure 6. Whole-genome sequencing identifies mutations that confer resistance to azole and FK506, as well as azole and geldanamycin.

(A) Sc-F-1 is resistant to azole and FK506 and cross-resistant to azole and cyclosporin A. (B) Resistance of Sc-F-1 is calcineurin-independent. Deletion of CNB1, which encodes the regulatory subunit of calcineurin required for its activation, does not affect resistance of Sc-F-1. (C) Deletion of MOT3 in the ancestral strain confers resistance to azole and FK506 equivalent to Sc-F-1, which is consistent with the MOT3^G265* allele of Sc-F-1 conferring resistance to azole and FK506. (D) Sc-G-13 is slightly resistant to azole and geldanamycin. (E) Resistance to azole and geldanamycin in Sc-G-13 is reduced when PDR^P865R is deleted and PDR1 is expressed on a plasmid. Resistance assays were performed and analyzed as in Figure 2, with incubation for 2 days at 30°C in YPD (A–D) or SD (E). CsA = cyclosporin A; GdA = geldanamycin; RAD = radicicol; and FL = fluconazole.

Figure 7. Six C. albicans lineages evolved with azole and FK506 share the same cross-resistance profile, and a mutation in CNA1 and LCB1 confers resistance.

(A) Each C. albicans lineage is resistant to high concentrations of FK506 or cyclosporin A in the presence of azole. (B) CNA1 ^S401* confers resistance to azole and calcineurin inhibitors in Ca-F-9. The C1201A mutation in CNA1, the gene encoding the catalytic subunit of calcineurin, leads to a premature stop codon and removal of the autoinhibitory domain. Deletion of CNA1 ^S401* in Ca-F-9 abrogates resistance, while deletion of one allele of CNA1 in the parental strain has no impact on sensitivity. (C) The A1169T mutation in orf19.6438 resulting in non-synonymous substitution (L390F) in this ortholog of S. cerevisiae LCB1 likely confers resistance in Ca-F-8. Lcb1 encodes a component of serine palmitoyltransferase that is responsible for the first committed step in sphingolipid biosynthesis, along with Lcb2. Inhibition of Lcb1 and Lcb2 with myriocin (900 nM) abrogates resistance of Ca-F-8. Resistance assays were performed and analyzed is in Figure 2, with incubation for 2 days at 30°C in YPD. GdA = geldanamycin; CsA = cyclosporin A; and FL = fluconazole.

Figure 8. Aneuploidies identified in four C. albicans lineages that evolved resistance to the combination of azoles and calcineurin inhibitors.

The genomes of six evolved strains were sequenced and profiled for copy number variants using CNV-Seq. Four of the strains contain aneuploidies: Ca-F-4, Ca-F-5, Ca-F-6, and Ca-F-7. Notably, chromosome 4 is increased in copy number in all four strains, suggesting that a locus on this chromosome is related to the mechanism of resistance. Blue = log₂ values; Red = moving average values.