Aneuploidy enables adaptation to brefeldin A in Candida albicans

Abstract

Candida albicans is the most prevalent opportunistic fungal pathogen. Both in vivo and in vitro studies have demonstrated that genome plasticity is a hallmark of C. albicans. While aneuploidy formation is a well-documented adaptive mechanism under various stress conditions, the response to brefeldin A-a compound that induces endoplasmic reticulum stress-remains poorly understood. In this study, we demonstrate that C. albicans adapts to subinhibitory and inhibitory concentrations of brefeldin A, primarily through the formation of chromosome 3 trisomy. These aneuploid strains were found to be unstable, reverting to euploidy in the absence of stress, accompanied by a loss of brefeldin A tolerance. We identified at least two genes on chromosome 3, SEC7 and CDR1, that contribute to this adaptive response. Notably, higher concentrations of brefeldin A selected for strains with increasingly complex aneuploidies. Our findings underscore the remarkable genomic plasticity of C. albicans and reveal aneuploidy as a reversible mechanism for adapting to brefeldin A stress. This study provides new insights into the role of aneuploidy in fungal adaptation and offers potential implications for understanding drug resistance mechanisms in pathogenic fungi.

Keywords: Candida albicans; ER stress; aneuploidy; brefeldin A; genome instability.

Figure 1

Susceptibility of SC5314 to brefeldin A. In growth curve assay (A), approximately 2.5 × 10³ cells/mL of SC5314 were grown in 150 μL of YPD broth with or without BFA. Growth was monitored at 30°C using a plate reader (Infinite F200 PRO; Tecan, Switzerland) at 15-min time intervals for 24 (h) Data are represented as the mean ± SD of three biological replicates. In the spot assay (B), cell densities were adjusted to 1 × 10⁷ cells/mL, and 3 µL of 10-fold serial dilutions were spotted onto YPD agar plates supplemented with BFA. Plates were incubated at 37°C for 48 h and then photographed. ^** p-values < 0.001, as determined by two-tailed Student’s t-test.

Figure 2

Exposure to a low amount of brefeldin A selects tolerant aneuploid adaptors. Approximately 2.5 × 10³ cells/mL of SC5314 were grown in 1.5 mL of YPD broth with or without 4 μg/mL of BFA. After 24 h of growth, the cultures were washed, diluted, and plated on YPD. A total of 120 colonies were randomly selected by spot assay on YPD plates supplemented with 32 μg/mL of BFA (A). Magenta circles indicate the parent strain, and cyan circles indicate the tolerant adaptors. In (B), the karyotypes of two BFA-tolerant adaptors were visualized using Ymap (Abbey et al., 2014). Read depth (normalized to that of the diploid parent) is shown on the y-axis on a log₂ scale converted to absolute copy numbers (1–4). Allelic ratios (A:B) are color-coded as follows: gray, 1:1 (A/B); cyan, 1:0 (A or A/A); magenta, 0:1 (B or B/B); purple, 1:2 (A/B/B); and blue, 2:1 (A/A/B).

Figure 3

Aneuploidy enabled adaptation to a high amount of brefeldin A. In (A), approximately 1 million cells of SC5314 were spread on a YPD plate or YPD plate supplemented with 32 μg/mL of BFA. The plates were incubated at 37°C for 3 days and then photographed. Eighteen adaptors (No. 1 to No. 18) were randomly selected and tested by spot assay on YPD plates supplemented with 32 and 64 μg/mL of BFA (B). In (C), karyotypes of all 18 adaptors were visualized using Ymap. The number of adaptors bearing the same karyotypes was indicated in the figure.

Figure 4

Chr3 genes associated with brefeldin A tolerance. Strains with heterozygous deletion of SEC7 and both heterozygous and homozygous deletions of CDR1 and CDR2 were assessed for BFA tolerance in comparison to the wild-type strain SC5314. Cells were spotted on TPD agar plates supplemented with BFA and incubated at 30°C for 48 h before being photographed.

Figure 5

Instability of aneuploids. (A) Four aneuploid strains with distinct karyotypes were daily passaged in YPD broth. Tolerance to BFA before and after passaging was tested by spot assay. (B) After passage, the adaptors were sequenced. Karyotypes before passing were shown on the left panel.

Figure 6

Chr3x3 drives genomic instability. One Chr3x3 strain (top) was spread on a YPD plate supplemented with 128 μg/mL of BFA. Eighteen adaptors were randomly selected and sequenced (bottom). These adaptors had 15 distinct karyotypes. The number of adaptors sharing the same karyotype is indicated on the left.

Figure 7

Chr5 and Chr6 genes are associated with BFA resistance. The wild-type control strain SC5314 and homozygous deletion mutants of MRR1, MRR2, TAC1, and MDR1 were tested for resistance to BFA. Strains with homozygous deletions of CDR1 and CDR2, as well as a heterozygous SEC7 deletion, were included as controls. Tenfold serial dilutions of each strain were spotted onto YPD plates containing the indicated concentrations of BFA and incubated at 37°C for 48 h.