Effects of fluconazole on the metabolomic profile of Candida albicans

Little is known about the effects of fluconazole on the metabolism of Candida albicans. We performed LC/MS-based metabolomic profiling of the response of C. albicans cells to increasing doses of fluconazole. C. albicans cells were cultured to mid-logarithmic growth phase in liquid medium and then inoculated in replicate on to nitrocellulose filters under vacuum filtration. Organisms were cultured to mid-logarithmic growth phase and treated with 0-4 mg/L fluconazole. Following metabolic quenching at mid-logarithmic growth phase, intracellular metabolites were extracted and analysed by LC/MS. Changes in pool sizes of individual metabolites were verified by Student's t-test, adjusted for multiple hypothesis testing by Benjamini-Hochberg correction. Distribution of metabolites was analysed by the Kyoto Encyclopedia of Genes and Genomes metabolic pathways database. We reproducibly detected 64 metabolites whose identities were confirmed by comparison against a pure standard and a library of accurate mass-retention time pairs. These 64 metabolites were broadly representative of eukaryotic central metabolic pathways. Among them 12 had their mean abundance significantly altered in response to increasing fluconazole concentrations. Pool sizes of four intermediates of central carbon metabolism (alpha-ketoglutarate, glucose-6-phosphate, phenylpyruvate and ribose-5-phosphate) and mevalonate were increased by 0.5-1.5-fold (PaEuroSa parts per thousand currency signaEuroS0.05). Five amino acids (glycine, proline, tryptophan, aminoisobutanoate and asparagine) and guanine were decreased by 0.5-0.75-fold (PaEuroSa parts per thousand currency signaEuroS0.05). Fluconazole treatment of C. albicans resulted in increased central carbon and decreased amino acid synthesis intermediates, suggesting a rerouting of metabolic pathways. The function of these metabolomic changes remains to be elucidated; however, they may represent previously unrecognized mechanisms of metabolic injury induced by fluconazole against C. albicans.