We found that tetrandrine (TET) can reverse the resistance of Candida albicans to fluconazole (FLC) and that this interaction is associated with the inhibition of drug efflux pumps. Mitochondrial aerobic respiration, which plays a major role in C. albicans metabolism, is the primary source of ATP for cellular processes, including the activation of efflux pumps. However, it was unclear if TET exerts its synergistic action against C. albicans via its impact on the mitochondrial aerobic respiratory metabolism. To investigate this mechanism, we examined the impact of FLC in the presence or absence of TET on two C. albicans strains obtained from a single parental source (FLC-sensitive strain CA-1 and FLC-resistant strain CA-16). We analysed key measures of energy generation and conversion, including the activity of respiration chain complexes I and III (CI and CIII), ATP synthase (CV) activity, and the generation of reactive oxygen species (ROS), and studied intracellular ATP levels and the mitochondrial membrane potential (DY m ), which has a critical impact on energy transport. Mitochondrial morphology was observed by confocal microscopy. Our functional analyses revealed that, compared with strains treated only with FLC, TET+FLC increased the ATP levels and decreased DY m in CA-1, but decreased ATP levels and increased DY m in CA-16 (P,0.05). Additionally, CI, CIII and CV activity decreased by 23-48 %. The production of ROS increased by two-to threefold and mitochondrial morphology was altered in both strains. Our data suggested that TET impacted mitochondrial aerobic respiratory metabolism by influencing the generation and transport of ATP, reducing the utilization of ATP, and resulting in the inhibition of drug efflux pump activity. This activity contributed to the synergistic action of TET on FLC against C. albicans.

Fermentation and aerobic respiration in mitochondria are coordinately regulated and compensated either when C. albicans grows in vitro or in the hosts, and the creature gain the strong viability. It’s insufficient to influent the growth, reproduction and pathogenicity of C. albicans by inhibiting the electron transport chain (ECT) CI, CII, CIII, CV, or fermentation related gene ADH1. Our study showed that the induction of AA (inhibitor of complex III) rather than SHAM (alternative oxidase inhibitor) abolishes the mitochondrial function completely （96% less ATP generation, 59% reduction in MMP), and increases ROS production significantly in ADH1-deleted mutant ( adh1Δ/ adh1Δ ) that in turn becomes hypersensitive to azole and apoptosis, less viable and more difficult to form hyphae. At the same time, the expression of virulence related genes ALS3 and HWP1 were significantly lower than that of WT under AA induction. Under the induction of AA, the mitochondrial function of WT was slightly damaged and cell apoptosis increased slightly，ROS production and sensitivity of azoles increased significantly, but mycelium formation and the growth of cells were not affected. Under aerobic growth, we observed an ADH1 - dependent mitochondrial effect in C. albicans demonstrated by 64% less ATP generation, 58% reduction in MMP and significant elevations of the ROS and apoptosis in ADH1 -deleted mutant. However, mycelium formation and azole susceptibility are not affected. Our results suggested that ADH1 plus CIII played an important role in antifungal activity by damaging mitochondrial function, inhibiting cell growth and hyphae formation, promoting apoptosis and reducing pathogenicity.