Combined Transcriptome and Metabolome Analysis Reveals That the Potent Antifungal Pyrylium Salt Inhibits Mitochondrial Complex I in Candida albicans

Based on the structural modification of SM21, xy12, a new pyrylium salt derivative with enhanced antifungal activities, was synthesized. The MICs (MIC₉₀) of xy12 against Candida albicans ranged from 0.125 to 0.25 μg/mL, about 2-fold lower than those of SM21. In addition, xy12 inhibited hypha and biofilm formation in C. albicans in a dose-dependent manner. A total of 3,454 differentially expressed genes and 260 differential metabolites were identified in the xy12-treated C. albicans by RNA-seq and non-targeted metabolomics. By integrating KEGG pathway enrichment analysis, we found that inhibition of oxidative phosphorylation was the important antifungal mechanism of action of xy12. Electron transport through mitochondrial respiratory complexes I to IV is the common process of oxidative phosphorylation. Compared with the sensitivity of the wild-type SC5314 to xy12, decreased sensitivities in mitochondrial complex I (CI)-deficient mutants and increased sensitivities in mitochondrial complex III- and IV-deficient mutants suggested that the antifungal effects of xy12 were dependent on CI. Consistently, xy12 exhibited antagonism with rotenone, an inhibitor of CI, and significantly inhibited the expression and activity of CI. Meanwhile, the phenotypes in the xy12-treated C. albicans were similar to those in the CI-deficient mutants, such as decreased ATP production, reduced mitochondrial membrane potential, loss of mitochondrial DNA, inability to utilize nonfermentative carbon sources, and decreased cell wall N-linked mannoproteins. Collectively, our results revealed that the pyrylium salt xy12 could constrain oxidative phosphorylation by inhibiting mitochondrial complex I in C. albicans, providing a novel lead compound for the development of mitochondria-targeted antifungal drugs. IMPORTANCE The development of new antifungal drugs is critical for solving the problem of antifungal resistance and expanding the limited variety of clinical antifungal drugs. Based on the modification of the pyrylium salt SM21, a new lead compound, xy12, was synthesized which was effective against Candida species both in vitro and in vivo. In this study, conjoined analysis of the transcriptome and metabolome elucidated the antifungal mechanism of action of xy12, which inhibited the activity of mitochondrial complex I in C. albicans. Targeting fungi-specific mitochondrial complex proteins has been reported as a promising antifungal strategy. Our study provided a new lead compound for targeting C. albicans mitochondrial complex I, which could be beneficial for discovering novel antifungal drugs.