Our study showed that Shikonin (SK) could provide an action against almost all Candida albicans isolates tested. More importantly, to some Fluconazole (FCZ)-resistant Candida albicans, the action of SK (MIC 80 value 4 µg/mL) was shown to be >16 times higher than that of FCZ (MIC 80 >64 µg/mL). To clarify the mechanism underlying this action, we performed a comparative study in untreated control C. albicans and C. albicans treated with SK. In this study, we found that SK treatment increased generation of endogenous reactive oxygen species (ROS) and decreased mitochondrial membrane potential. Furthermore, anti-oxidants N-acetylcysteine (NAC) and glutathione (GSH) could reduce the antifungal activity of SK significantly in C. albicans. Our analyses also identified 9 differentially expressed genes, which were related to glycolysis-related genes (CDC19 and HXK2), fermentation-related genes (ALD5 and ADH1), antioxidant defense-related genes (SOD2 and SOD5), thioredoxin reductase-related gene (TRR1), mitochondrial respiratory electron transport chain-related gene (MRF1) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidoreductase-related gene (EBP1). These results suggest that mitochondrial aerobic respiration shift and endogenous ROS augmentation contribute to the action of SK against C. albicans. Key words Candida albicans; shikonin; reactive oxygen species; mitochondrial membrane potentialCandida albicans, the major opportunistic fungal pathogen of humans, normally exist as commensal. However, in patients with an impaired immune system such as those suffering from acquired immunodeficiency syndrome or cancer, C. albicans can cause recurrent superficial infection and fatal deep-seated and disseminated candidiasis. 1-4)Treatment for C. albicans includes antifungal drugs, mainly the azoles. Unfortunately, with the increasing clinical use of the azoles, azoles-resistant isolates are occurring more frequently. 5,6) Thus the development of new, effective antifungal agents is strongly needed in medicine.Shikonin (SK) (Fig. 1) is set in the orient, originally in China. This molecule is the major constituent of the red pigment extracts from the roots of the plant Lithospermum erythrorhizon SIEB. et ZUCC (LE). SK is widely used as a material to prepare an ointment called "Shiun-ko" which is used to treat wounds, burn and hemorrhoids in Japan. 7,8) Our study found that SK exhibit significant antifungal activities almost all C. albicans isolates tested. More importantly, to some Fluconazole (FCZ)-resistant C. albicans, the action of SK minimum inhibitory concentration (MIC 80 value 4 µg/mL) was shown to be >16 times higher than that of FCZ (MIC 80 >64 µg/mL). And the antifungal properties may relate to SKmediated oxidative damage and breakdown of mitochondrial membrane potential. Antifungal Susceptibility Testing Assays were performed on 10 isolates of C. albicans according to methods of the CLSI (formerly NCCLS) (M27-A). MATERIALS AND METHODS Strains and Agents9-11) The initial concentration of the fungal sus...

Amphotericin B (AmB) is a polyene antifungal drug widely used for systemic fungal infections. In this study, a metabonomic method using gas chromatography-mass spectrometry (GC/MS) was developed to characterize the metabolic profiles of Candida albicans cells exposed to AmB. Thirty-one differentially produced metabolites between AmB-treated and the control groups were identified, among which 10 metabolites were upregulated and 21 metabolites were downregulated. These differentially produced metabolites were mainly involved in polyamines synthesis, tricarboxylic acid (TCA) cycle, oxidative stress, glutathione metabolism, lipid synthesis and glycolysis. Further experiments showed that the polyamines including putrescine, spermidine, and spermine played an important role in the sensitivity of C. albicans cells upon AmB treatment, and combined use of AmB and inhibitors of polyamine biosynthesis pathway might be a potential antifungal strategy. This study provided a systemic view of the metabolic pattern in C. albicans upon exposure to AmB, which shed new light on the mechanisms of action of antifungal agents.

Amphotericin B (AmB) is a polyene antibiotic produced by Streptomyces nodosus and has been used for >50 years in the treatment of acute systemic fungal infections. In the present study, we demonstrated that lysine, an essential amino acid, could enhance the effect of AmB against Candida albicans in vitro, although lysine itself did not exert a fungicidal effect. In addition, the combination of AmB with lysine could provide an enhanced action against Candida parapsilosis and Cryptococcus neoformans compared with AmB alone. Lysine could also enhance the antifungal effect of caspofungin or nystatin. An enhanced effect of the combination of lysine with AmB was observed for the prevention of biofilm and hypha formation. Furthermore, our results demonstrated that lysine-mediated oxidative damage, such as the generation of endogenous reactive oxygen species, may be the mechanism underlying the enhancing effect of lysine on AmB. Our results also showed that CaMCA1 gene plays an important role in increasing the sensitivity of C. albicans cells upon AmB treatment. Using AmB together with lysine may be a promising strategy for the therapy of disseminated candidiasis.