Selection of Ethanol Tolerant Strains ofCandida albicansby Repeated Ethanol Exposure Results in Strains with Reduced Susceptibility to Fluconazole

Candida albicansis a commensal yeast that has important impacts on host metabolism and immune function, and can establish life-threatening infections in immunocompromised individuals. Previously,C. albicanscolonization has been shown to contribute to the progression and severity of alcoholic liver disease. However, relatively little is known about howC. albicansresponds to changing environmental conditions in the GI tract of individuals with alcohol use disorder, namely repeated exposure to ethanol. In this study, we repeatedly exposedC. albicansto high concentrations (10% vol/vol) of ethanol—a concentration that can be observed in the upper GI tract of humans following consumption of alcohol. Following this repeated exposure protocol, ethanol small colony (Esc) variants ofC. albicansisolated from these populations exhibited increased ethanol tolerance, altered transcriptional responses to ethanol, and cross-resistance/tolerance to the frontline antifungal fluconazole. These Esc strains exhibited chromosomal copy number variations and carried polymorphisms in genes previously associated with the acquisition of fluconazole resistance during human infection. This study identifies a selective pressure that can result in evolution of fluconazole tolerance and resistance without previous exposure to the drug. Overall design: Cultures of SC5314 or Esc strains (small colony strains isolated from populations repeatedly exposed to ethanol) were grown to post-exponential phase and 10^8 cells for each were seeded into 1 mL cultures of DMEM with 10% H2O or EtOH and incubated for 4 hours at 37°C with 5% CO2. Immediately following the incubation, cells were centrifuged at 6000 rpm for 1 min and supernatant was removed. Cells were then resuspended in 1 mL of RNA-later and immediately frozen at -80°C until they were ready for extraction. Cells were then thawed on ice. Cells were lysed and RNA was extracted by bead-beating. RNA was isolated using the Invitrogen Purelink RNA mini kit with the on-column DNase treatment. RNA was eluted in 40uL of ultrapure H2O and RNA yield and purity were assessed on a Nanodrop. Three biological replicates of SC5314, Esc6, Esc7, or Esc8 following exposure to medium A with 10% water or 10% EtOH were sent for sequencing. All samples sequenced had RNA integrity scores of 9.2 or greater. cDNA libraries were made by Genewiz and RNA-seq was performed by Genewiz using a HiSeq platform: gene expression was considered significant only if adjusted-P-value (calculated by Benjamini-Hochberg test) was less than 0.05 and log2-fold change was greater than the absolute value of 0.5. RNA-sequencing was aligned to Candida albicans SC5314 (assembly ASM18296v3) as the reference genome. Raw counts were determined using DESeq. Submitter states that missing raw files are due to file loss.