Chromosome 5 monosomy of Candida albicans controls susceptibility to various toxic agents, including major antifungals

Abstract

Candida albicans is a prevailing fungal pathogen with a diploid genome that can adapt to environmental stresses by losing or gaining an entire chromosome or a large portion of a chromosome. We have previously found that the loss of one copy of chromosome 5 (Ch5) allows for adaptation to the toxic sugar l-sorbose. l-Sorbose is similar to caspofungin and other antifungals from the echinocandins class, in that it represses synthesis of cell wall glucan in fungi. Here, we extended the study of the phenotypes controlled by Ch5 copy number. We examined 57 strains, either disomic or monosomic for Ch5 and representing five different genetic backgrounds, and found that the monosomy of Ch5 causes elevated levels of chitin and repressed levels of 1,3-β-glucan components of the cell wall, as well as diminished cellular ergosterol. Increased deposition of chitin in the cell wall could be explained, at least partially, by a 2-fold downregulation of CHT2 on the monosomic Ch5 that encodes chitinase and a 1.5-fold upregulation of CHS7 on Ch1 that encodes the protein required for wild-type chitin synthase III activity. Other important outcomes of Ch5 monosomy consist of susceptibility changes to agents representing four major classes of antifungals. Susceptibility to caspofungin increased or decreased and susceptibility to 5-fluorocytosine decreased, whereas susceptibility to fluconazole and amphotericin B increased. Our results suggest that Ch5 monosomy represents an unrecognized C. albicans regulatory strategy that impinges on multiple stress response pathways.

Fig 1

Products of PCR amplification obtained with total DNA of each strain and primers specific for the MTLa or MTLα locus. Presented are parental strains RO29R4, JRCT1, RO20G1, and SC5314 and their mutants, as indicated. Also presented are control amplifications with 3153A and its representative mutant Sor41, monosomic for Ch5 (2), as well as its exceptional mutant, Sor5, that became resistant to sorbose due to a large deletion on one of two Ch5 copies (36). Note that two bands, the upper band for MTLα on Ch5b and the lower band for MTLa on Ch5a, are amplified from DNA of parental strains or the exceptional Sor5, while one band of either kind is amplified from the mutants. See Table 2 for the relationships between strains.

Fig 2

DNA profiling of the monosomic Ch5 by tiling aCGH. The aCGH log₂ of the derivative/parental ratio for each of the tiling probes on each chromosome is plotted according to the chromosomal position of the probe. Thus, every point of the plot corresponds to a probe. The horizontal line at position 0 on the ordinate corresponds to no DNA change. Note the global diminution of the DNA on Ch5b but not on the other chromosomes in the derivatives Sor91, Sor321, Sor322, and Sor343 (Table 2).

Fig 3

Examples of susceptibility of the mutants with monosomic Ch5 to antifungal agents compared to parental strains. Shown is the spot assay for growth on control YPD medium as well as on YPD medium in the presence of CAS, 5FC, FLU, or AmB. Drug concentrations are indicated. From left to right, 10⁴, 10³, 10², and 10¹ cells were spotted on each plate and incubated at 37°C for up to 8 days. Control YPD plates were incubated for 2 days. Strains are indicated on the left. Names of the derivatives are shown under the names of the corresponding parentals, RO29R4, JRCT1, RO20G1, SC5314, and 3153A (Table 2). The monosomy, 1n, or disomy, 2n, condition of Ch5 is indicated. Larger papillae are interpreted as arising from mutations compensating for growth, as these appeared on supplemented as well as control media. Note that Ch5 monosomic mutants demonstrated less growth and smaller colonies than their normal parents on the control YPD medium. However, this pattern is reversed in the presence of toxic agent CAS or 5FC or enhanced in the presence of toxic agent FLU or AmB.

Fig 4

Susceptibilities of the mutants with monosomic Ch5 to cell wall-damaging agents compared to parental strains. Shown is the spot assay for growth on control YPD medium as well as on YPD medium in the presence of caffeine, SDS, hygromycin B, and Congo red. Drug concentrations are indicated. From left to right, 10⁴, 10³, 10², and 10¹ cells were spotted on each plate and incubated at 37°C for 2 to 5 days. Control YPD plates were incubated for 2 days. Strains are indicated on the left (Table 2). The monosomy, 1n, or disomy, 2n, of Ch5 is indicated.

Fig 5

Survival of strains monosomic or disomic for Ch5 in the presence of zymolyase. Ch5 monosomic or disomic strains are indicated with continuous or dotted lines, respectively. The control derivatives of 3153A carrying various chromosome alterations are indicated with dashed lines. Ch5 ploidy is also indicated. The following monosomic mutants are included: Sor321, Sor322, Sor91, Sor92, Sor93, Sor71, Sor72, Sor73, Sor122, Sor123, Sor41, Sor43, Sor1210(60), Sor126, Sor128, and Sor125(55). The following reduplicated strains are included: Sor321-R, Sor91-R, Sor92-R, Sor93-R, Sor71-R, Sor72-R, Sor73-R, Sor125Sor125(55)-R, Sor122-R, and Sor123-R. The following control derivatives of 3153A are included: Sor5, m2, m5, m6, m7, m8, m15, and m16. The following parental strains are included: RO29R4, JRCT1, SC5314, and 3153A. See Table 2 for the relationships between strains. Each data point for monosomic mutants, reduplicated strains, or parentals is averaged from three independent experiments. Each data point for control derivatives of 3153A is averaged from two independent experiments.