In previous studies, replicate populations evolved in the presence of FLC, and their gene expression was profiled (2
); these expression data were used in comparisons with strains that evolved AmB resistance in this study to identify consensus changes. All populations initially subjected to a high concentration (256 μg/ml) became extinct, and no AmB resistance evolved (data not shown); this is unlike resistance to FLC, in which wild types subjected to a high concentration (256 μg/ml) initially did survive and evolved resistance. Here, five different populations were evolved in the presence of AmB under two different regimens, long (Fig. ) and short (Fig. ). Under both regimens, cell division kept pace with the repeated dilution of cultures, and no extinctions occurred. Under the long regimen, the MIC50
remained equal to or greater than the ambient AmB concentration. Under the short regimen, the MIC remained equal to or less than the ambient AmB concentration. In all five populations from the long and short regimens, in which the mass culture and three randomly selected single-colony isolates were assayed, the final MIC50
of AmB was 128 or 256 μg/ml.
FIG. 1. Evolution of resistance measured as MIC50 in three replicate yeast populations over 1,100 generations, during which the concentration of AmB was doubled every 100 generations. Circles, AmB concentration in the medium; diamonds, triangles, and squares, (more ...)
FIG. 2. Evolution of resistance measured as MIC50 in two replicate populations over 57 transfers, during which the concentration of AmB was doubled every other transfer. Circles, AmB concentration in the medium; diamonds, triangles, and squares, MICs of the two (more ...)
Next, a representative of each AmB-resistant population was assayed for alterations in patterns of gene expression in the absence of any drug. Figure depicts the consensus genes whose levels of expression were altered 1.5-fold greater or lesser in each of the five populations. Numerous other changes were also detected among each of the five AmB-resistant strains; the individual genes and their expression values are listed in Table S1 in the supplemental material. The numbers of persistently overexpressed genes identified by our criteria ranged between 172 and 316 among the five AmB-resistant strains; of these examples of overexpression, between 43 and 64% were unique to the respective strains. Similarly, the numbers of underexpressed genes identified ranged from 84 to 139 among the five strains; of these, between 49 and 70% were unique to the respective strains.
FIG. 3. Gene expression in the five evolved populations resistant to AmB measured in the absence of the drug. Replicate bar graphs facing upwards represent increased levels of expression relative to that of the ancestor. Graphs with bars facing downward represent (more ...)
Next, we searched for genes whose levels of expression were stably altered with evolved resistance to AmB in this study and to FLC in a previously reported study (3
). Six genes were overexpressed in all five AmB-resistant strains from this study and in the three FLC-resistant strains that arose in one regimen of selection for FLC resistance in the earlier study (3
). In this regimen, the concentration of FLC increased in four increments from 16 μg/ml to 256 μg/ml over 400 generations, and each resistant type carried a dominant mutation in the regulatory gene PDR1
. The other regimen was based on a single exposure to a high concentration of FLC, and each of three independently evolved resistant strains had a loss of function in ERG3
. No genes were overexpressed in all five AmB- and six FLC-resistant strains from the two studies.
Each of the six genes identified above is known to be involved either with multidrug resistance or with cellular stress: YGR035C is a putative gene whose transcription is activated by transcription factors Yrm1p and Yrr1p along with genes associated with multidrug resistance (15
is a plasma membrane ABC transporter involved in drug resistance (9
is a lysophosphatidic acid acyltransferase gene responsible for enhanced phospholipid synthesis during organic solvent stress and for tolerance of calcofluor white (12
is a 3-methylbutanal reductase and NADPH-dependent methylglyoxal reductase gene that is stress induced (5
is a phosphatidylinositol transfer gene controlled by the multiple-drug-resistance regulator Pdr1p that localizes to lipid particles and microsomes and controls levels of various lipids (20
), and YPL088 is a putative aryl alcohol dehydrogenase gene whose transcription, like those of YGR035C and other genes, is activated by the paralogous transcription factors Yrm1p and Yrr1p (15
Does the expression of any of these six nonessential genes contribute to the capacity to evolve drug resistance? The next experiments used the null mutants of each gene in an otherwise wild-type and FLC-sensitive background to initiate short-term evolution experiments in 32 μg/ml or 128 μg/ml of FLC, a concentration for which resistance is known to evolve rapidly in wild-type populations (3
), even those of very small sizes. It was not practical in this study to assay the ability to evolve resistance to AmB because of the much longer time required for resistance to appear in populations subjected to sublethal concentrations of this agent. In these experiments with FLC, cell density invariably decreased in 32 μg/ml or 128 μg/ml FLC, but not in the absence of FLC, over several days. Evidence of adaptation was (i) the recovery of cell density to levels comparable to those in the absence of drug, which occurred in some but not all replicates, and (ii) an MIC50
of FLC of 128 or 256 μg/ml in those that did recover at the end of the growth period. The wild-type, ancestral strain always achieves resistance under these conditions. Similarly, three of the deletion strains achieved resistance in all replicate populations under both concentrations of FLC. Three additional deletion strains, the pdr16
Δ, and hsp90
Δ strains, were tested in this way; all these strains consistently evolved resistance using this test (data not shown). Although the nature of resistance was not determined in these cases, these deletion strains might achieve resistance in the following ways. The pdr1
Δ strain may accumulate gain-of-function mutations in PDR3
, a transcriptional regulatory gene similar to PDR1
. Although defective in efflux of FLC, the pdr5
Δ strain might achieve resistance through alterations in sterol metabolism even though it is especially sensitive to FLC (2
). Last, it is known that resistance via gain-of-function mutations in the relevant transcriptional regulators is not sensitive to conditions of low levels of Hsp90 (6
Those deletion strains in which extinctions or near extinctions occurred, the yor1Δ, ict1Δ, and pdr16Δ strains, were selected as candidates for the next round of experiments, in which each deletion strain was competed against the wild-type ancestor over 108 generations in the presence of 0, 32, and 128 μg/ml FLC. Here, populations were completely dependent on the occurrence of mutations for resistance to avoid extinction. The criterion for “winning” the competition was not the initial fitness but rather the capacity for FLC-resistant mutations to arise and propagate in the population. Under these conditions, the mean fitness for each marked strain can be calculated over the entire experiment based on initial and final densities for each transfer as the total number of doublings.
As a control, G418-resistant and NAT-resistant versions of the wild-type progenitor were competed with one another (Table ). As expected, there was no consistent bias in the resistant types appearing in the absence of FLC (mean fitness of G418 resistance compared to NAT resistance over 108 generations of 0.998 with 0.018 standard deviations [SD]; n = 3 measurements). Surprisingly, in FLC, there were strong marker effects on adaptation. In 32 μg/ml FLC, NAT resistance was consistently favored (mean fitness of G418 resistance compared to NAT resistance over 108 generations of 0.956 with 0.003 SD; n = 3 measurements), and in 128 μg/ml, G418 resistance was consistently favored (mean fitness of G418 resistance compared to NAT resistance over 108 generations of 1.024 with 0.004 SD; n = 3 measurements). These marker effects, while small in terms of fitness per generation, had a strong effect on final proportions after 108 generations of evolution and were therefore taken into account in interpreting the outcome of the remaining competitive adaptation experiments. The mechanism by which these marker effects are conditional regarding the FLC concentration is unknown.
Mean fitness of test strains marked with G418 resistance compared to that of the progenitor marked with NAT resistance over 108 generations of direct competition
The YOR1 deletion strain showed a clear growth disadvantage relative to the wild type in the absence of FLC (Table ). The yor1Δ strain was also among the minority after 108 generations in each concentration of FLC but did not become extinct in any of the replicates. Of the minority YOR1 deletion types surviving 108 generations in FLC, all representatives tested were resistant. In 128 μg/ml FLC, the predominance of the wild type marked by NAT resistance was opposite to the direction of the marker effect in which G418 resistance is at an advantage. Under both FLC concentrations, the proportion of the YOR1 deletion strain roughly paralleled the growth disadvantage of this strain in the absence of FLC. Even with a severe reduction in population size in the YOR1 deletion populations, the ability to acquire resistance to FLC was not impaired.
The ICT1 deletion strain showed mixed effects. There was little or no growth disadvantage of the deletion strain relative to the ancestor in the absence of FLC. The ict1Δ strain showed mixed results at 32 μg/ml, probably indicating rough parity with the wild type in adaptive potential. Unexpectedly, however, the ict1Δ strain showed a clear evolutionary advantage over the wild type in 128 μg/ml. Like the deletion of YOR1, the deletion of ICT1 does not impede the evolution of resistance. Unlike the deletion of YOR1, however, the deletion of ICT1 actually enhances the ability of populations to acquire FLC resistance relative to the wild type.
The PDR16 deletion strain showed the most consistent results in the competitive adaptation experiments. The pdr16Δ strain showed a marked advantage over the wild type in the absence of FLC. Under both concentrations of FLC, the pdr16Δ strain was driven to extinction in all replicates. In 128 μg/ml FLC, this extinction is opposite of the direction of the marker effect in which G418 resistance is favored. At 32 μg/ml, the extinction of the pdr16Δ strain exceeded the expected marker effect where NAT resistance is favored over G418 resistance but not to the point of extinction. Despite its ability to evolve resistance to FLC in some replicate populations when grown alone (Fig. ), the pdr16Δ strain is at a clear disadvantage to the wild type in a competition.
FIG. 4. Evolution of FLC resistance in deletion strains for the six genes overexpressed in the AmB resistance strains from this study and in the three FLC-resistant strains reported previously by Anderson et al. (3) evolved through gradual increases in the FLC (more ...)
The rationale for this study was to test whether downstream gene expression changes found after resistance appears might actually be necessary for the appearance and/or maintenance of a resistant phenotype. The PDR16
deletion showed the greatest impairment of the ability of populations to evolve resistance to FLC. PDR16
is involved in lipid metabolism and is under the regulation of the PDR1
transcriptional network. The deletion of PDR16
is already known to confer increased drug susceptibility (20
), although here, it was no more susceptible to FLC than the wild type. In explaining the evolutionary outcomes with the PDR16
deletion, population size and evolutionary potential are always intertwined, as larger populations have a larger supply of mutations, some of which confer greater fitness and result in a greater chance for the population to escape extinction by spreading rapidly to a high frequency in the presence of the drug. In the evolutionary competitions, however, small population size alone cannot explain the observed deficit of the pdr16
Δ strain in its ability to adapt to the presence of FLC; when grown alone in the presence of FLC, one replicate population even went to apparent extinction only to develop resistance and then abruptly recover in size. The most likely explanation is that the PDR16
deletion somehow reduces the capacity of a population to maintain a resistant state in the presence of FLC. FLC is known to cause membrane stress by creating a deficiency of ergosterol. The best general test of this hypothesis would be to reduce or eliminate the expression of PDR16
of an already resistant strain in the presence of FLC. More specifically, the loss of PDR16
expression may alter membrane characteristics such that the function of the relevant ABC transporters is impaired, influx rates are increased (20
), or membranes are made less robust to changes in sterol content. Each of these three possibilities is amenable to experimental testing.
This study shows that the effect of the deletion of PDR16
extends beyond drug susceptibility and actually reduces the evolutionary potential to develop FLC resistance. The effect of the PDR16
deletion may extend to other drugs or conditions conferring membrane stress. PDR16
may play a similar role in the clinically important pathogen Candida albicans
, in which it is part of the transcriptional network regulating the ABC transporters commonly involved in resistance to FLC. A potential application of this finding is that PDR16
could be considered as a cotarget for the prevention of resistance evolution (1
). The general principle of cotargets tested here could also be extended to other drugs, genes, pathogens, and forms of resistance.