The evolution of drug resistance threatens human health worldwide. An emerging strategy to mitigate drug resistance is combination therapy. The fate of multidrug-resistant pathogens depends on their fitness relative to susceptible counterparts, yet the fitness consequences of multidrug resistance remain enigmatic. Here, we dissect fitness consequences of the evolution of resistance to antifungal drug combinations in the leading human fungal pathogen, Candida albicans. We focus on the most widely deployed antifungals, the azoles, and inhibitors of the molecular chaperone Hsp90 and protein phosphatase calcineurin, which regulate cellular stress responses required for azole resistance. We find tradeoffs such that adaptation to drug combinations is associated with reduced fitness in distinct environments, including those relevant to the human host. We identify mutations associated with fitness tradeoffs in clinical isolates and that influence morphogenesis, a key virulence trait. Thus, we delineate evolutionary constraints that may minimize the evolution of resistance to antifungal combinations.

Several questions emerge about the fitness consequences of resistance to drug combinations. Is resistance to drug combinations costly in the absence of drug or in the presence of a single drug? Does resistance to these drug combinations, particularly ones that target stress response regulators, confer cross-resistance to novel stress environments? Or does resistance to drug combinations create a trade-off in other environments? To address these questions, we take advantage of two distinct sets of strains of FL-resistant C. albicans that evolved resistance to FL and an inhibitor of Hsp90 or calcineurin in either a human host (in vivo) or a laboratory environment (in vitro). Here, we determine the fitness effects of resistance to drug combinations in these two sets of C. albicans lineages and identify mutations associated with fitness trade-offs in clinical isolates. Resistance to the combination of azole and an inhibitor of Hsp90 or calcineurin could in principle evolve by the accumulation of mutations that confer resistance to each class of drug, as was the case with most of the experimental evolution lineages. Alternatively, azole-resistance mechanisms that are independent of the cellular stress response regulators could evolve, as was the case with the clinical isolates that evolved resistance in the human host. Leveraging whole genome sequence data, we identified alleles that render azole resistance independent of Hsp90 and calcineurin in the clinical isolates: TAC1 A736V , UPC2 A643V , and ERG11 R467K . For both the in vitro and in vivo evolved strains, adaptation to these drug combinations was associated with fitness trade-offs in distinct stress environments relevant to host conditions, including survival in host immune cells. Additionally, while most strains that evolved resistance to the drug combinations retained the capacity to filament in response to inducing cues, a key virulence trait, clinical isolates recovered late during treatment and harboring the TAC1 A736V allele were unable to filament in response to Hsp90 inhibitors. This work illuminates evolutionary constraints that may forecast reduced vulnerability of antifungal drug combinations to the evolution of resistance in pathogen populations.

Drug-combination therapy is a promising strategy to decrease the rate of drug resistance. Combination therapy can enhance the utility of antimicrobials by hindering the evolution of resistance, such as by targeting cellular processes required for resistance. An example is to cripple cellular stress response regulators when treating human pathogens like C. albicans. C. albicans is the fourth most common cause of hospital-acquired infection, and systemic C. albicans infections are recalcitrant to treatment, with mortality rates approaching 40% (). The most frequently deployed class of antifungals is the azoles; however, they are fungistatic and vulnerable to resistance (). Resistance is often contingent on the molecular chaperone Hsp90, which regulates key stress response proteins, including the protein phosphatase calcineurin (). Combination therapy with an inhibitor of Hsp90 or calcineurin and an azole provides a powerful strategy for rendering azole-resistant C. albicans infections responsive to treatment (). However, we must anticipate that resistance to drug combinations will evolve. We recently established an experimental evolution approach to track the evolution of resistance to drug combinations, and while most lineages went extinct, a minority was able to evolve resistance to the azole fluconazole (FL) in combination with the Hsp90 inhibitor geldanamycin (GdA) or the calcineurin inhibitor FK506. The fitness consequences of the evolution of resistance to drug combinations remain enigmatic.

Drug-resistance mechanisms provide a clear fitness advantage for the pathogen in the presence of drug, but their maintenance in the absence of drug selection is contingent on minimal fitness costs of resistance (). Resistance mechanisms often alter key cellular functions; thus, resistance is frequently associated with a cost in the absence of drug. Epidemiological models and experimental evidence suggest that this can slow the spread of resistance (), yet drug resistance is not necessarily costly. For example, an isochromosome (i5L) in the fungal pathogen Candida albicans confers resistance to the antifungal azoles and is not deleterious in the absence of drug (). Understanding whether drug resistance is costly in clinically relevant environments is crucial to evaluating whether resistance will persist.

The evolution of drug resistance is a looming public health crisis. Imprudent use of antimicrobials in agriculture and medicine compromises the effectiveness of our arsenal of drugs, as the rate at which new antimicrobials are developed does not keep pace with the rapid emergence of resistance (). Resistance complicates medical outcomes, imposes significant financial burdens, and threatens a post-antibiotic era of medicine (). Thus, reducing the spread of drug resistance is of critical importance.

There are two alternative explanations for the absence of filamentation in late-stage clinical isolates: Hsp90 inhibition no longer induces filamentation in these strains, or GdA does not inhibit Hsp90 function in these strains. To test whether genetic reduction of HSP90 levels induces filamentation in these strains, we monitored filamentation in response to tetracycline-mediated transcriptional repression of HSP90 in the earliest and last clinical isolate backgrounds. Transcriptional repression of HSP90 in the first and last clinical isolates was sufficient to induce filamentation ( Figure 7 A), confirming that Hsp90 represses filamentation in these strains. A possible cause for this inability to respond to GdA is that it could be effluxed from the cell. Consistent with this possibility, the last two isolates have increased mRNA levels of CDR1 and CDR2, ABC transporters that are transcriptionally regulated by TAC1 (). To determine whether TAC1blocks morphogenesis in response to GdA, we replaced a native allele of TAC1 with TAC1in the earliest clinical isolate background. Unlike the parental strain, the strain expressing TAC1did not filament in response to GdA ( Figure 7 B), suggesting that TAC1leads to the overexpression of pumps sufficient to efflux GdA.

(B) TAC1 A736V confers resistance to GdA. After 6 hr in YPD with 10 μM GdA at 30°C, CaCi-2 grows filamentously, while CaCi-17 grows as yeast. The ability to undergo morphogenesis is abolished in CaCi-2 when a WT copy of TAC1 is replaced by TAC1 A736V in that background. Scale bar represents 20 μm.

(A) Transcriptional repression of HSP90 in both the first and last clinical isolate induces filamentation. All strains were grown in YPD with a low concentration of doxycycline (0.03125 μg/ml) for 18 hr at 30°C, for transcriptional repression of HSP90 in the tetO- HSP90 strains.

We next examined the ability of evolved lineages to undergo morphogenesis in response to filament-inducing cues. Under standard laboratory conditions, C. albicans grows as yeast, but in response to specific cues, it transitions into a filamentous form. The ability to transition between these morphologies is a key C. albicans virulence trait (), and thus another measure of fitness. Filamentation can be induced by inhibition of Hsp90, by elevated temperature of 39°C that overwhelms Hsp90 function due to global protein misfolding, or by exposure to serum at 37°C (). We monitored filamentation of the in vitro and in vivo evolved lineages in response to these cues. C. albicans lineages evolved in vitro filamented readily in response to serum and 39°C ( Figure 6 A). These strains are hypersensitive to GdA, such that both standard filament-inducing concentrations (10 μM) and low concentrations (2.5 μM) impaired survival ( Figure S3 ). Lineages evolved in vivo were also able to filament in response to serum and 39°C. Intriguingly, while isolates recovered from the patient early during treatment filamented in response to GdA, the last two isolates recovered did not ( Figure 6 B).

(B) Clinical isolates grow as yeast in 30°C, but filament in response to 39°C and 10% serum at 37°C. Late-stage isolates are unable to filament in the response to 10 μM GdA at 30°C, unlike early-stage isolates. Strains were grown in YPD for 6 hr. Scale bar represents 10 μm.

(A) In vitro evolved strains grow as yeast in 30°C, but filament in response to 39°C and 10% serum at 37°C in YPD after 6 hr.

The fitness deficit of many evolved strains in Hsuggests that they may be more susceptible to killing by host immune cells such as macrophages. We tested this directly by quantifying macrophage killing for each evolved and ancestral strain. J774A.1 macrophages were inoculated with individual C. albicans ancestral and evolved lineages at an MOI of 1:1. After 1 hr, macrophages were lysed and C. albicans survival was determined. Lineages that had lower fitness in Halso had reduced survival in macrophages ( Figures 5 and S2 ). Indeed, later clinical isolates and most aneuploid in vitro evolved lineages had lower survival than their ancestors, suggesting that strains resistant to antifungal drug combinations would be less fit in a host than their ancestors. We examined this by performing competition experiments of the evolved strains relative to a marked version of the ancestral strains in the host model system Galleria mellonella. These wax moth larvae are a well-established system for the study of fungal pathogenesis (). We observed substantial variation between larvae, suggesting an early bottleneck during infection ( Figure S2 ), as has been observed with mouse models of candidiasis (). Although many evolved strains showed a trend toward decreased fitness, only the in vitro evolved strain LCB1had a significant decrease in fitness in the invertebrate host model.

Strains were incubated with J774A.1 macrophages for 1 hr before macrophages were lysed and viable C. albicans cells quantified by plating for CFUs. Differences between strains were determined by ANOVAs with Bonferroni corrections for multiple comparisons and subsequent Tukey post hoc tests. Data represented as mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01.

Clustering in vitro-evolved strains indicated that point mutations in the drug targets CNA1 and HSP90 had little fitness consequence overall and that the two aneuploid strains, Chr4,5R,6,7(2n+1) and Chr4,R(2n+1), were the least fit ( Figure 4 H). Clustering in vivo evolved strains indicated that late clinical isolates had very similar phenotypes ( Figure 4 I).

Like the in vitro evolved strains, the later clinical isolates generally fared worse than or equal to the earliest isolate in the different stress environments ( Figure 4 ), with heat stress as a notable exception. While in vitro evolved strains fared worse at high temperature, later clinical isolates showed increased fitness. This may be due to the selective pressure of febrile temperatures that accompany infections in the host.

In general, the in vitro-evolved strains performed worse than or equal to the ancestor in novel stressful environments, suggesting that resistance to FL and GdA or FL and FK506 does not promote cross-resistance to other environmental stresses ( Figure 4 ). The strain that often fared the worst had the most aneuploidies (Chr4,5R,6,7(2n+1)), while the strains with point mutations were largely no different in fitness from the ancestor (with the exception of LCB1in CFW). The fitness deficit of evolved strains was greatest in H, a reactive oxygen species produced by neutrophils and macrophages upon ingestion of foreign particles like invading microbes, suggesting that these strains might not fare well in the host.

While there is generally a cost of resistance to drug combinations in the absence of drug, resistance to drug combinations may confer cross-resistance to different stressful environments. Alternatively, drug combination resistance mechanisms may be costly in stressful environments, unlike the environments the strains were evolved in (antagonistic pleiotropy). We sought to identify whether resistance to drug combinations has a fitness disadvantage in a variety of host relevant, novel stress environments. Fitness was measured during exposure to cell wall stress (calcofluor white [CFW]), cell membrane stress (SDS), salt stress (NaCl), oxidative stress (hydrogen peroxide [H]), and temperature stress (42°C; Figures 4 C–4G).

While strong selective pressure for resistance is exerted in the presence of the drug combination, resistance mechanisms may be costly when selection is alleviated in the absence of drugs (). We determined whether strains resistant to FL and GdA or FL and FK506 had a fitness disadvantage in the absence of drug (rich medium alone, yeast extract peptone dextrose [YPD]), by competing them against a GFP-marked version of the ancestral strain. Frequently, a cost of resistance to drug combinations was found ( Figure 4 A). Four of seven in vitro evolved strains were less fit than the ancestor in the absence of drug, as were four of five in vivo evolved strains. We also tested whether resistance to drug combinations is costly in the presence of FL alone, which the ancestor is resistant to. While two in vitro evolved strains were significantly less fit than the ancestor in FL, one strain was more fit (LCB1), and the remaining strains were not significantly different from the ancestor ( Figure 4 B). In contrast, there is a clear trend of increasing fitness with each subsequent clinical isolate in the presence of FL.

(A–G) Fitness relative to the ancestor was measured for in vitro and in vivo evolved lineages by competitive fitness assays in (A) the absence of drug, (B) FL alone, (C) the cell wall stress CFW, (D) the cell membrane stress SDS, (E) salt stress, (F) the oxidative stress H 2 O 2 , and (G) heat stress. Each circle represents the mean of three replicate competition experiments, with standard error bars. Differences in fitness between strains were determined by ANOVAs with Bonferroni corrections for multiple comparisons and subsequent Tukey post hoc tests. Data represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01.

To determine whether these alleles confer Hsp90- and calcineurin-independent azole resistance, we replaced the native UPC2, ERG11, and TAC1 alleles in the first clinical isolate, CaCi-2, with the mutant alleles and assayed resistance to FL alone and FL in combination with GdA or FK506 ( Figure 3 ). All strains were able to grow to a high concentration of FL alone, as expected. Single copies of ERG11, UPC2, and TAC1individually had minimal effect on resistance to FL and GdA and caused a minor increase in resistance to FL and FK506. However, reconstituting the genotype of the last clinical isolate (CaCi-17) with respect to the alleles of interest (UPC2/UPC2; ERG11/ ERG11; TAC1/ TAC1) in the background of CaCi-2 conferred nearly the same degree of resistance to FL and GdA or FL and FK506 as is found in CaCi-17 ( Figure 3 ), suggesting that these alleles are largely responsible for Hsp90- and calcineurin-independent azole resistance.

(A–C) TAC1 A736V , UPC2 A643V , and ERG11 R467K were placed in the background of CaCi-2 individually or in combinations that reflect genotypes found in later clinical isolates concurrent with increases in resistance to drug combinations (UPC2 A643V /UPC2 ERG11 R467K /ERG11 R467K in CaCi-13; UPC2 A643V /UPC2 ERG11 R467K /ERG11 R467K TAC1 A736V /TAC1 in CaCi-17). Resistance to FL was assessed as a single agent (A) or in combination with GdA (B) or FK506 (C). Individual mutations confer a modest increase in resistance to FL and FK506, although no effect on resistance to FL and GdA. Recapitulating the genotype of the last clinical isolate, CaCi-17, confers resistance to FL and GdA and FL and FK506 to nearly the same FL concentration as found in CaCi-17. Susceptibility assays performed in duplicate in YPD at 37°C for 48 hr. Optical densities were standardized to no drug control wells. TAC1 ∗ = TAC1 A736V ; UPC2∗ = UPC2 A643V ; ERG11∗ = ERG11 R467K .

In order to identify mutations conferring the two-step increase in Hsp90- and calcineurin-independent FL resistance, we leveraged high-coverage whole-genome sequence for each of the isolates, which was validated by strong concordance between Illumina base calls and Sequenom iPlex genotyping assays (personal communication, D. Thompson and A. Regev, Broad Institute of MIT and Harvard; see the Supplemental Experimental Procedures and Table S1 ). The first small increase in resistance is accompanied by heterozygous UPC2and homozygous ERG11(at CaCi-13; see Figure 1 and Table S1 ); heterozygous TAC1arose coincident with the second, larger increase in resistance (at CaCi-16). Only four nonsynonymous substitutions occurred in coding regions accompanying the first increase in resistance, and of those, UPC2(a regulator of ergosterol biosynthesis genes) and ERG11(the target of FL) have established roles in azole resistance (). TAC1(a transcriptional activator of ABC drug transporters and a known mechanism of azole resistance;) is the single nonsynonymous substitution that accompanied the larger increase in resistance to the drug combinations.

In contrast, deleting CNA1 in the background of most in vitro evolved strains abrogates resistance to FL ( Figure 2 C), indicating that the mechanism of resistance in these strains is calcineurin dependent. However, deletion of CNA1 in the background of the strain with LCB1does not abrogate FL resistance, indicating that this resistance mechanism is calcineurin independent.

Despite not having been exposed to GdA or FK506, late-stage clinical isolates evolved resistance to these drugs in combination with FL. One possible explanation is that late-stage isolates acquired a FL resistance mechanism that is independent of both Hsp90 and calcineurin. To test this, we constructed strains in the background of the first (CaCi-2) and last (CaCi-17) clinical isolates where the only copy of HSP90 was placed under the control of a tetracycline-repressible promoter (tetO-HSP90/hsp90). A low concentration of the tetracycline analog doxycycline abrogated FL resistance of the early clinical isolate, but only modestly reduced resistance of the late clinical isolate ( Figure 2 A), phenocopying the effect of GdA. To determine whether the late clinical isolate is resistant to FL independent of calcineurin, we assayed FL resistance in strains where the catalytic subunit of calcineurin (CNA1) was deleted in the background of CaCi-2 and CaCi-17. As with Hsp90 depletion, deletion of calcineurin in CaCi-2 abrogated FL resistance while calcineurin deletion in CaCi-17 only modestly reduced resistance ( Figure 2 B), phenocopying resistance to FL and FK506 in CaCi-2 and CaCi-17. These results indicate that resistance to FL is largely dependent on Hsp90 and calcineurin in the early clinical isolate but independent from these regulators in the late clinical isolate.

(C) Deleting CNA1 in the background of four in vitro-evolved strains abrogates FL resistance, in contrast to the clinical isolates. Deleting CNA1 in the background of LCB1 L390F does not, however, indicating that FL resistance in this strain is calcineurin independent. Susceptibility assays performed in duplicate in YPD at 37°C for 48 hr (A and B) or at 30°C for 24 hr (C). Optical densities were standardized to no drug control wells.

(B) Rendering calcineurin nonfunctional by deletion of its catalytic subunit, CNA1, abrogates FL resistance in CaCi-2 but has only a minor effect on FL resistance in CaCi-17.

The Mechanism of Resistance in Late-Stage Clinical Isolates Is Largely Independent of Hsp90 and Calcineurin, in Contrast to Most Experimentally Evolved Strains

Intriguingly, the clinical isolates evolved a two-step increase in resistance to FL in combination with GdA or FK506 despite having no prior exposure to those inhibitors. This two-step increase in resistance is reflected in a two-step increase in fitness relative to the ancestor in either FL and GdA or FL and FK506 ( Figure 1 D). The fitness profiles correspond to differences in individual growth rates ( Figure S1 ).

The increase in resistance to drug combinations in experimentally evolved strains and clinical isolates suggests that they may have a competitive advantage over their ancestors in these environments. Indeed, competitive fitness assays of each evolved strain relative to a marked version of the ancestor revealed that strains evolved in FL and FK506 are more fit than the ancestor in that environment, while the strain evolved in FL and GdA has no advantage in that drug combination ( Figure 1 C). Similarly, the strain evolved in FL and GdA is more fit than the ancestor in FL and GdA, while all but one strain evolved in FL and FK506 are not ( Figure 1 C). To a large extent, competitive fitness corresponds to differences in growth measured in isolation ( Figure S1 ).

The second set of strains resistant to FL and GdA or FK506 evolved resistance in a human host. Six strains from a series of C. albicans oral isolates from an HIV-infected individual sampled over a 2-year period of treatment with FL comprise this set (). These isolates are clonally related, representing a case of in vivo evolution. FL resistance of isolates recovered early during treatment is abrogated by GdA or FK506 ( Figure 1 B). Resistance to FL in combination with GdA or FK506 increases in isolates recovered later during treatment, despite the patient not having been exposed to GdA or FK506. The increase in resistance to the drug combinations occurs in two steps, and isolates reflecting these transitions were selected for subsequent fitness assays ( Figure 1 ).

Resistance to FL in combination with GdA or FK506 was studied in two sets of strains with distinct genetic backgrounds and different life histories, providing a broader view of the scope of fitness consequences that accompany the evolution of resistance to antifungal drug combinations. The first set was lineages that we previously evolved in FL and an inhibitor of Hsp90 (GdA) or calcineurin (FK506) ( Figure 1 A) (). Experimental evolution was initiated with a C. albicans erg3Δ/erg3Δ strain that is resistant to FL in a manner that depends on Hsp90 and calcineurin (). Mechanisms of resistance were subsequently identified in seven lineages, using either a hypothesis-driven approach or inferences from whole genome sequencing (). Resistance mechanisms were validated by allele swap assays. Four of the strains resistant to FL and FK506 harbor widespread aneuploidy (Chr4,5R,6,7(2n+1), Chr4,7(2n+1), Chr4(2n+1), Chr4,R(2n+1)), implicated in resistance. The remaining two strains resistant to FL and FK506 acquired a substitution in a sphingolipid biosynthesis regulator (LCB1) or a premature stop codon in the catalytic subunit of the drug target itself, calcineurin (CNA1). Resistance to FL and GdA occurred in one strain and was due to mutation in the drug target gene, HSP90 (HSP90).

(C and D) Fitness of in vitro (C) and in vivo (D) evolved strains was assessed relative to their respective ancestors in FL and GdA (top) and FL and FK506 (bottom). Generally, in vitro evolved strains passaged in FL and FK506 (Chr4(2n+1), Chr4,R(2n+1), Chr4,7(2n+1), Chr4,5R,6,7(2n+1), LCB1 L390F , CNA1 S401Stop ) are more fit in that environment but not in FL and GdA and vice versa (HSP90 D91Y was passaged in FL and GdA). In vivo-evolved strains increase in fitness in both FL and GdA and FL and FK506 over time, despite not having experienced these drug combinations in the host. Susceptibility assays were performed in duplicate at 30°C for 48 hr, where optical densities were standardized to no drug control wells.

(B) A series of clinical isolates recovered over 2 years from a single patient have a two-step increase in resistance to drug combinations. Subsequent analyses focus on key isolates reflecting the two-step increase in resistance to these drug combinations (CaCi-2, CaCi-12, CaCi-13, CaCi-15, CaCi-16, and CaCi-17), indicated by the pink triangles.

(A) Of the seven experimentally evolved strains, six are resistant to FL and FK506, and one is resistant to FL and GdA.

Discussion

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Cowen L.E. Genetic and genomic architecture of the evolution of resistance to antifungal drug combinations. Here we illuminate key fitness consequences of the evolution of resistance to drug combinations in C. albicans lineages that evolved resistance in vitro or in the human host. While both sets of strains evolve resistance from FL alone to FL in combination with GdA or FK506, they have very different life histories. One set is comprised of a series of clinical isolates recovered from a patient treated with FL over time, and the other set is experimentally evolved from a strain whose FL resistance is conferred by loss of function of Erg3, followed by selection with FL in combination with GdA or FK506 (). Strains evolved in test tubes versus in the human host experience vastly different environments; passage of C. albicans in a host model has been associated with 5-fold slower growth and greater phenotypic and genotypic diversity compared with passage in vitro (). Despite these differences and the distinct mechanisms of azole resistance () ( Figures 2 and 3 ), these strains share several fitness traits.

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2 O 2 ( Overall, the fitness profile of the clinical isolates is very similar to that of the in vitro evolved strains: competitive fitness is increased in evolved strains in FL, FL and GdA, and FL and FK506, while it is decreased in the alternative stresses SDS, CFW, NaCl, and H Figure 4 ). Furthermore, evolved strains were generally more susceptible to killing by macrophages than their ancestors ( Figure 5 ). Thus, adaptation to drug combinations is associated with trade-offs in terms of reduced fitness in host relevant environments. These fitness costs would reduce the persistence of resistant pathogens when drug selection is removed following termination of therapeutic regimens. While later clinical isolates are generally less fit than their progenitor, the deficit is not so great that they are eliminated by the host and their emergence could still be favored under conditions that select for Hsp90 or calcineurin-independent azole resistance, such as with febrile temperatures. Even if resistance were costly in the absence of drug, it could still evolve in response to selection.

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Berman J. A tetraploid intermediate precedes aneuploid formation in yeasts exposed to fluconazole. We also observed frequent fitness costs to aneuploidy, which is an established drug-resistance mechanism. Four of the in vitro evolved strains that are resistant to FL and FK506 are aneuploid, and three of the four strains exhibit fitness defects in most stressful environments and in the absence of drug ( Figure 4 ). Notably, the strain Chr4(2n+1), which has an additional copy of the small chromosome 4 alone, suffers minimal negative fitness consequences. In contrast, the strain with the most aneuploid chromosomes, Chr4,5R,6,7(2n+1), which carries an extra copy of nearly half of its genome, has a fitness disadvantage in almost every environment tested and often has the greatest magnitude of cost. This suggests that the burden of replicating extra chromosomes is costly in many environments. While aneuploidy in S. cerevisiae generally reduces growth in rich medium and in stressful conditions, it can confer a fitness advantage depending on karyotype and conditions (). Aneuploid Candida isolates are often recovered from patients, indicating that aneuploidy is maintained in a clinical context (). Both inhibition of Hsp90 and treatment with FL can induce aneuploidy (), suggesting that this could be a prevalent resistance mechanism to treatment with these drug combinations.

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Verstrepen K.J. Different levels of catabolite repression optimize growth in stable and variable environments. The fitness disadvantage exhibited by most strains in several novel stress environments indicates resistance to FL and GdA or FL and FK506 does not confer cross-resistance to these conditions. Altogether, our results indicate that resistance to drug combinations results in trade-offs in these novel conditions. This is consistent with previous findings of trade-offs in novel environments (). High costs of resistance can drastically diminish the likelihood of it evolving and being maintained, such as with resistance to the antifungal amphotericin B (). However, strains grown in variable environments can produce “generalists” that thrive in diverse conditions (), and given that the human host is a complex and variable environment, cross-resistance to host-related environments was a plausible expectation. Understanding the relationship between fitness trade-offs measured in vitro and those that manifest in the human host will require greater insight into fitness of pathogens in host model systems.

While the fitness profiles of in vitro and in vivo strains are quite similar, a notable difference is at elevated temperature ( Figures 4 and S1 ). At 42°C, the later clinical isolates outcompeted the early isolate. In contrast, the in vitro-evolved strains were equally or less fit than the ancestor at 42°C. A possible explanation for this discordance is that in the host fever may provide selective pressure for growth at higher temperatures. Febrile temperatures destabilize proteins, titrating Hsp90 away from clients to stabilize these proteins, and allow for high temperature growth. This reduction in available Hsp90 selects for azole resistance to evolve toward independence from Hsp90 and its client protein calcineurin in the clinical isolates. While high temperature provides selective pressure for azole resistance that is independent of Hsp90 and calcineurin, which results in resistance to drug combinations in clinical isolates, the converse is not true: resistance to drug combinations explicitly, as selected for in the in vitro evolved strains, does not confer resistance to high temperature.

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Wickens C.

Cowen L.E.

Kohn L.M. Mode of selection and experimental evolution of antifungal drug resistance in Saccharomyces cerevisiae. Resistance to an antifungal in combination with an Hsp90 or calcineurin inhibitor can arise by the evolution of an antifungal resistance mechanism that does not depend on the cellular stress responses regulated by Hsp90 or calcineurin, or it can arise by the evolution of resistance to the inhibitor of Hsp90 or calcineurin in an antifungal-resistant background. Resistance of the first clinical isolate is dependent on Hsp90 and calcineurin, but in the last clinical isolate, resistance is largely independent of these regulators ( Figure 2 ). In contrast, resistance to drug combinations in two of three in vitro evolved strains is still dependent on calcineurin. This highlights a difference between strains evolved with inhibitors of Hsp90 or calcineurin, as opposed to the selective pressures operating in the human host. The resistance mechanisms that prevail depend on the nature of the selection pressure, the initial genotype, and the accessibility of adaptive peaks in the adaptive landscape. Divergent adaptive trajectories could be favored by low fitness intermediates. Indeed, there is negative epistasis between an Hsp90-independent mechanism of resistance (a hyperactivating mutation in PDR1, a transcriptional activator of drug pumps) and an Hsp90-dependent mechanism of resistance (loss of function of Erg3) in S. cerevisiae (). The extent of epistasis between resistance mechanisms is a fascinating area for future study.

A736V imparts additional resistance to the drug combination and is an example of a mechanism of positive cross-resistance (resistance to more than one drug, here, FL and GdA) ( Palmer and Kishony, 2013 Palmer A.C.

Kishony R. Understanding, predicting and manipulating the genotypic evolution of antibiotic resistance. Within a strain, different traits can also vary in dependence on Hsp90 and calcineurin. This is illuminated by our findings that resistance to FL is largely independent of Hsp90 and calcineurin in late-stage clinical isolates, as opposed to a resulting from resistance to GdA or FK506. In contrast, morphogenesis remains under the control of Hsp90 in these strains, such that transcriptional repression of HSP90 induces filamentation despite the strains being blocked in morphological response to GdA ( Figure 7 ). While resistance to FL and GdA in the last clinical isolate is mostly independent from Hsp90, GdA efflux facilitated by TAC1imparts additional resistance to the drug combination and is an example of a mechanism of positive cross-resistance (resistance to more than one drug, here, FL and GdA) ().