Acquisition of aneuploidy provides increased fitness during the evolution of antifungal drug resistance

Abstract

The evolution of drug resistance is an important process that affects clinical outcomes. Resistance to fluconazole, the most widely used antifungal, is often associated with acquired aneuploidy. Here we provide a longitudinal study of the prevalence and dynamics of gross chromosomal rearrangements, including aneuploidy, in the presence and absence of fluconazole during a well-controlled in vitro evolution experiment using Candida albicans, the most prevalent human fungal pathogen. While no aneuploidy was detected in any of the no-drug control populations, in all fluconazole-treated populations analyzed an isochromosome 5L [i(5L)] appeared soon after drug exposure. This isochromosome was associated with increased fitness in the presence of drug and, over time, became fixed in independent populations. In two separate cases, larger supernumerary chromosomes composed of i(5L) attached to an intact chromosome or chromosome fragment formed during exposure to the drug. Other aneuploidies, particularly trisomies of the smaller chromosomes (Chr3-7), appeared throughout the evolution experiment, and the accumulation of multiple aneuploid chromosomes per cell coincided with the highest resistance to fluconazole. Unlike the case in many other organisms, some isolates carrying i(5L) exhibited improved fitness in the presence, as well as in the absence, of fluconazole. The early appearance of aneuploidy is consistent with a model in which C. albicans becomes more permissive of chromosome rearrangements and segregation defects in the presence of fluconazole.

Figure 1. Drug resistance profiles throughout evolution.

(A) Populations evolved with fluconazole (D9, D11, and D12) adapted to the drug and showed increased fluconazole MIC . Populations evolved without fluconazole (N1–N6) show no change in MIC and (B) maintain karyotype stability. Whole chromosomes were separated by CHEF, stained with Ethidium Bromide (Left) and (C) subjected to Southern hybridization with a CEN5 probe. Lane P, the progenitor strain karyotype.

Figure 2. Rapid acquisition of i(5L) occurred in all fluconazole treated populations.

(A–C) Whole chromosome CHEF gel analysis of populations D9, D11, and D12 at generations ∼3.3, 50, 140, 165, 200, 230, 260, 300, and 330. (D–F) Southern blot analysis of the CHEF gels using a CEN5 probe identified both Chr5 homologs as well as either independent i(5L) or att-i(5L) at generation 3.3 in all three populations. The i(5L) aneuploidy was detected in all subsequent generations of D9 and D11, while the att-i(5L) was lost in D12 after generation D12-260. The CEN5 probe also hybridized to a novel band at ∼1.5 Mb in population D9 (black arrow).

Figure 3. Clones at generation 3.3 with i(5L) have a significant fitness advantage in the presence of fluconazole.

(A) Karyotype analysis of five D9-3.3 single colonies by CHEF followed by (B) Southern hybridization to a CEN5 probe. Genetic heterogeneity and clonal interference exists: 3 clones appear diploid, one clone has i(5L), and one clone is homozygous for Chr5. (C) Fitness competitions were performed, as described in the Materials and Methods, either in the absence (white bars) or presence (black bars) of fluconazole. Each clone was competed against the same NAT1-marked progenitor strain and the average relative fitness was determined. Error bars represent the standard deviation of 3 replicate experiments. All three clones with i(5L) had significantly increased fitness in the presence of 0.5 µg/ml fluconazole (asterisks, paired t-test, p≤0.05), with no disadvantage in the absence of the drug.

Figure 4. Rapid acquisition of i(5L) coincided with multiple whole chromosome aneuploidies.

CGH analysis of single colonies from populations D9-3.3 and D11-3.3 found that clones were either completely diploid (A,D) and had lower fluconazole MICs, or were aneuploid and had higher MICs (B,C). Clone D9-3.3-D (B) contains i(5L) and is trisomic for Chrs 3, 4, and 7, while clone D11-3.3-A (C) contains multiple copies of i(5L) and is trisomic for Chrs 3 and 7.

Figure 5. Southern blot analysis of the ∼1.5 Mb SNC.

(A) Population D9-260 exhibited colony size variability on YPD plates. (B,C) CHEF/Southern analysis of single colonies detected a novel ∼1.5 Mb SNC in small (lanes a–e) but not in large (lanes f–i) colonies. The ∼1.5 Mb SNC hybridized to CEN5 and Chr3R probe CDR1 (arrows), but not to probes from probes further to the left on Chr3 (CDR2 and CEN3) or from Chr6. Both small and large colonies maintained independent i(5L) (arrowhead).

Figure 6. Supernumerary chromosome i(5L)-3R increases resistance to fluconazole.

(A) CGH analysis of small colony D9-260-a detected ∼4 additional copies of Chr5L and trisomy of Chr6 and Chr7, while (B) CGH of a large colony D9-260-F identified ∼2 additional copies of Chr5L (i(5L)) and Chr7 trisomy. E-test strip assays indicated that the smaller colony MIC (∼64 µg/ml) (A, insert) was higher than the larger colony MIC (∼8 µg/ml) (B, insert). (C) CGH analysis of the isolated ∼1.5 Mb SNC DNA from D9-260-b. Genes from Chr5L (breakpoint at CEN5) and Chr3R (breakpoint just to the left of CDR1) are highly overrepresented on the ∼1.5 Mb SNC. (D) Fitness assays, of D9-260 clones are consistent with slow growth of both the small (D9-260-a) and larger (D9-260-F) colony clones relative to the progenitor, suggesting that the population has accumulated a large number of mutations. Nonetheless, small colony clones containing the ∼1.5 Mb SNC were slightly more fit in the presence of fluconazole (black bars, ∼128 µg/ml, twice the MIC of the experimental population D9-260) than clones that did not carry the SNC. Fitness of the aneuploid strains was not reduced significantly in the absence of fluconazole (white bars). Asterisks indicate fitness levels that are significantly different from the progenitor strain.

Figure 7. Changes in aneuploid chromosomes are common during adaptation to fluconazole.

Population D12 underwent drastic changes in aneuploid chromosome number, which indicates that clonal interference was high, and coincides with a fluctuation in fluconazole MIC. (A) D12-165 has att-i(5L) and is trisomic for Chr7, (B) D12-260 lost the att-i(5L) and gained Chr4 trisomy, and (C) D12-330 lost the Chr7 trisomy, but maintained the Chr4 trisomy. (D) Population D12-165 was passaged for an additional ∼100 generations in the absence of fluconazole and it lost the att-i(5L) aneuploidy, but maintained the Chr7 trisomy.

Figure 8. Transformation of att-i5L results in loss of i(5L) and retention of the intact Chr5 portion.

(A) Strain D12-165 contains an att-i(5L) chromosome with two CEN5 structures (top) and one normal Chr5 homolog (bottom). Transformation was used to disrupt orf19.1963 on Chr5L with the NAT1 marker. There are 4 existing orf19.1963, three on the att-i(5L) (I, II, III) and one on the full-length Chr5 homolog (IV). (B) CHEF/Southern blot analysis of 13 NAT1 positive transformants indicates that in all but 3 cases the NAT1 insertion occurred on the att-i(5L) at any locus I-III. In the remaining 3 transformants, NAT1 insertion occurred at locus III and coincided with detachment of the i(5L) and reformation of the independent Chr5 homolog. This same phenomenon was observed during the original D12 evolution experiment.