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-␤-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.

Chromosome 5 of Human Pathogen Candida albicans Carries Multiple Genes for Negative Control of Caspofungin and Anidulafungin Susceptibility

“…CHT2, CSU51, or PGA4 controls the decrease of 1,3-␤-glucan and increase of chitin in the cell wall. Caspofungin tolerance is associated with cell wall remodeling, including the decrease of 1,3-␤-glucan and increase of chitin (4,12,30). Thus, we asked whether the same is true for the CHT2, CSU51, or PGA4 deletion strains (see Materials and Methods for the assays).…”

“…For this purpose, we prepared a sequential series of genetically related strains including parental strain JRCT1 followed by Ch5 monosomic mutant JMC200-3-3 followed by Ch5 reduplicated derivative JMC200-3-3R (F. Yang and E. Rustchenko, unpublished data). The construction of a series of matched strains with a copy number of Ch5 alternating between two and one is a well-established procedure in our laboratory, as described in detail previously (4,29). Briefly, a Ch5 monosomic mutant was obtained by plating cells of JRCT1 on YPD medium supplemented with a lethal amount of caspofungin, whereas the Ch5 reduplicated derivative was obtained due to spontaneous duplication of the monosomic Ch5 when growing cells of JMC200-3-3 on YPD solid medium in the absence of selection.…”

“…The same amount of cells from each strain was prepared. The 1,3-␤-glucan levels were determined by the aniline blue assay, as previously described (4,24). Briefly, 500 l of cell suspensions from each strain was resuspended with 100 l of 6 M NaOH, followed by incubation at 80°C for 30 min.…”

“…The chitin content was determined by measuring the absorbance of glucosamine released by acid hydrolysis of the purified cell wall as described previously (4,25). Briefly, approximately 3 ϫ 10 3 CFU was plated on YPD plates and incubated at 37°C.…”

“…The expression of at least two such genes, CSU51 (orf19.1105.2) and CSU53 (orf19.3931), is finely tuned by antisense regulation (6). Recently, we used laboratory mutants to demonstrate that the reversible loss of Ch5 also controls tolerance to the major echinocandin caspofungin, such that strains with one copy acquire caspofungin tolerance whereas strains that spontaneously duplicate monosomic Ch5 revert to susceptibility (4). Based on the model system of sorbose resistance, Ch5 can carry multiple genes encoding negative regulators of echinocandin susceptibility.…”