Cryptococcus neoformans overcomes stress of azole drugs by formation of disomy in specific multiple chromosomes

Abstract

Cryptococcus neoformans is a haploid environmental organism and the major cause of fungal meningoencephalitis in AIDS patients. Fluconazole (FLC), a triazole, is widely used for the maintenance therapy of cryptococcosis. Heteroresistance to FLC, an adaptive mode of azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the resistance was unknown. We used comparative genome hybridization and quantitative real-time PCR in order to show that C. neoformans adapts to high concentrations of FLC by duplication of multiple chromosomes. Formation of disomic chromosomes in response to FLC stress was observed in both serotype A and D strains. Strains that adapted to FLC concentrations higher than their minimal inhibitory concentration (MIC) contained disomies of chromosome 1 and stepwise exposure to even higher drug concentrations induced additional duplications of several other specific chromosomes. The number of disomic chromosomes in each resistant strain directly correlated with the concentration of FLC tolerated by each strain. Upon removal of the drug pressure, strains that had adapted to high concentrations of FLC returned to their original level of susceptibility by initially losing the extra copy of chromosome 1 followed by loss of the extra copies of the remaining disomic chromosomes. The duplication of chromosome 1 was closely associated with two of its resident genes: ERG11, the target of FLC and AFR1, the major transporter of azoles in C. neoformans. This adaptive mechanism in C. neoformans may play an important role in FLC therapy failure of cryptococcosis leading to relapse during azole maintenance therapy.

Figure 1. FLC E-tests.

Approximately 1×10⁶ cells of each strain were plated on YPD media and E-test strips were placed on the media. The plates were incubated at 30°C for 72 h. Strains: H99 (wild type), H99^R64 (resistant at 64 µg/ml of FLC), H99^Rvt16, and H99^Rvt26 (H99^R64 derivatives obtained by daily transfer of H99^R64 on drug-free media for 16 and 26 days, respectively).

Figure 2. Chromosome 1 and 4 are duplicated in H99^R64.

(A) Gene expression profiles. Gene expression patterns of H99^R64 were compared to those of the wild type strain, H99. A total of eight arrays including three biological repeats and dye-reversed sets were performed as described in materials and methods. Each column (lane 1 to 8) represents a microarray experiment and each row represents the expression of a gene on the array arranged by its chromosomal position. A total of 4149 significant genes were plotted after SAM analysis with a mean FDR of 5%. Chromosomes where upregulated genes are clustered are indicated. The relative expression levels are represented by color as shown in the bar. (B) CGH plot of H99^R64. The genomic DNA of the experimental strain was hybridized against the genomic DNA of the reference strain, H99. Each panel represents the CGH plot of each chromosome. Chromosome number is indicated in the right side corner of each panel. The x-axis represents the position of each gene arranged in the order of its chromosomal location. The y-axis plots gene copy number as a running average over seven genes calculated from the log₂ ratio of relative hybridization intensity. (C) Copy number of four genes on Chr1 determined by qPCR. Four probes (chr1A, chr1B, chr1C, and chr1D) at different locations on Chr1 were compared to two control probes; one located on Chr3 (chr3A) and the other on Chr11 (chr11A) in H99 and H99^R64, respectively. (D) Copy number of Chr4 genes (chr4A and chr4B) was compared to that of the probe (chr3A) on the endogenous control Chr3 in H99 and H99^R64.

Figure 3. Gain and loss of FLC resistance positively correlates with the number of chromosomes duplicated.

(A) CGH plots of Chr1, 4, and 3 for strains H99^R64, H99^Rvt16, and H99^Rvt26. (B) CGH plots of Chr1, 4, 10, 14, and 3 for strains H99^R32, H99^64L, and H99^R128. (C) Copy number of four genes in H99-derived strains with different levels of resistance. The relative gene copy number of four probes (chr1A, chr4A, chr10A and chr14A) located on Chr1, 4, 10, and 14 was compared to the control probe (chr3A) on Chr3. The same genomic DNA from the strains used for CGH arrays was used for qPCR assays. Only the chromosomes involved in duplication are shown. Chr3 serves as a non-duplicated chromosome control.

Figure 4. Gene duplication determined by colony qPCR.

Gene dosage was determined by qPCR in four independent colonies of H99 derived strains by stepwise selection on YPD agar plates containing increasing concentrations of FLC: (A) 32 µg/ml; (B) 64 µg/ml; (C) 128 µg/ml. The relative gene copy number of four probes (chr1A, chr4A, chr10A and chr14A) located on Chr1, 4, 10, and 14 was compared to the control probe on Chr3 (chr3A). The 0 passage samples represent initial colonies that appeared 4 days after plating the parental strains on plates containing FLC. The gene duplication in those colonies was also determined following 4 and 8 passages on agar media with the same concentration of FLC.

Figure 5. The correlation between ERG11 location and the phenotype.

The diagram on the right represents the chromosomal location of ERG11 and its corresponding phenotype is shown on the left (FLC E-tests). The position of 4 tested genes (chr1A, chr1B, chr1C, and chr1D) on Chr1 is indicated at the top of the diagram.

Figure 6. The importance of ERG11 and AFR1 in Chr1 duplication.

(A) Gene duplication patterns in C1345 derived strains. Gene copy number was determined by qPCR in resistant colonies derived from C1345 which contains two copies of ERG11, one on Chr1 and the other on Chr3. Two different colonies resistant to FLC 32, 64 and 128 µg/ml are shown respectively. Gene copy number was quantified by measuring four probes (ERG11 = chr1A, chr1D, chr3A and chr4B) located on Chr1, 3 and 4, respectively, in comparison to the control probe (chr5A) on Chr5. (B) CGH plots of Chr1, 3, and 4 for C1345^R64 and C1345^R128 that tolerate 64 and 128 µg/ml FLC, respectively. Only the chromosomes involved in duplication are shown. (C) Gene copy number was determined by qPCR in resistant colonies derived from three independent transformants (C1347, C1348, and C1350), each with a single copy of ERG11 on Chr3. Strains resistant to 32 and 64 µg/ml FLC are shown. The probes located on Chr1, 3, and 4 were compared to the control probe (chr5A) on Chr5. (D) Heat map of CGH results: lanes 1–3 represent the CGH array of three independent strains, C1347, C1348, and C1350, resistant at 32 and 64 µg/ml FLC, respectively. The relative copy number is represented by color as shown in the bar. Red indicates that the copy number of the genes is close to 2. (E) CGH plots of Chr1, 4, 5, 9, 10, and 3 for the C1371 (afr1Δ ^R1) strain resistant at 1 µg/ml of FLC.