| Multidrug resistance aggravates treatment failure of invasive infections with Candida albicans (C. albicans), a most common Candida pathogen. Our previous study showed that concomitant use of berberine (BBR) and fluconazole (FLC) provided a synergistic action against FLC-resistant C. albicans clinical strains in vitro. To clarify the mechanism underlying this action, we performed a comparative proteomic study in untreated control cells and cells treated with FLC and/or BBR in 2 clinical strains of C. albicans resistant to FLC (0304103, 01010) and the results were confirmed by real-time RT-PCR. Our analyses identified 16 differentially expressed proteins, most of which were related to energy metabolisms (e.g., Gap1, Adh1, and Aco1). Functional analyses revealed that FLC+BBR treatment increased mitochondrial membrane potential, decreased intracellular ATP level, inhibited ATP-synthase activity, and increased generation of endogenous reactive oxygen species (ROS) in FLC-resistant strains. In addition, checkerboard microdilution assay showed that addition of anti-oxidant ascorbic acid or reduced glutathione reduced the synergistic antifungal activity of FLC+BBR significantly. These results suggest that mitochondrial aerobic respiration shift and endogenous ROS augmentation contribute to the synergistic action of FLC + BBR against FLC-resistant C. albicans.To further reveal the specific synergistic mechanism of the combination of FLC and BBR against FLC-resistant C. albicans clinical strains, in this study, we performed a cDNA microarray analysis in untreated control cells and cells treated with FLC and/or BBR in 3 clinical strains of C. albicans resistant to FLC (0304103, 01010 and 632) and the results were confirmed by real-time RT-PCR. By taking overlap of differentially expressed genes identified in 3 clinical strains treated with or without FLC and/or BBR, our analyses identified 70 differentially expressed genes, 26 of which were function-unknown genes; 35 of which were related to multiple biochemical functions such as transcription regulator activity, translation regulator activity, RNA bingding, protein binding, cell cycle, cellular homeostasis, organelle organization, filamentous growth, transporter activity and multiple enzyme activity. After bioinformatics analysis and reading pertinent literature, we found that these function-known genes differentially expressed after the combnation treatment of FLC + BBR were mainly involved in 3 kinds of biochemical function: energy metabolisms, DNA damage and vacuolation. The expressed tendency of energy metabolisms-related genes were consistent with the results of the proteomic study performed previously. According the cDNA chip results, we speculated that the final mechanism to kill the Candida cells with the combination treatment of FLC + BBR is the DNA damage caused by BBR and there might be a natural BBR-resistant mechanism in C. albicans by storing BBR up in the vacuole.To explore the key mechanism of the synergistic action of FLC + BBR and to find the potential"ex-ROS"mechanism, based on the results of the proteomic and cDNA chip studies performed previously, we observed the location of BBR in C. albicans cells by uesing electron microscope and laser confocal microscopy. The results showed that the BBR was stored up in the vacuole and the vacuole were magnified after BBR treatment. In addition, further experiments including spot assay, checkerboard microdilution assay and real-time RT-PCR were performed. Based on the results of these experiments, we raised the hypothesis that C. albicans cells could store BBR up in the vacuole according to the"vesicle trafficing"mechanism, which caused the BBR-resistance in C. albicans. When combined with FLC to against C. albicans, BBR could released from the vacuole by the vacuolar membrane-disruptive effect of FLC and the cells were killed by the augmentation of ROS and DNA damage. This research provides a new direction to study multidrug-resistance in C. albicans. |
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