Farnesol-Induced Apoptosis in <em>Candida albicans</em> Is Mediated by Cdr1-p Extrusion and Depletion of Intracellular Glutathione

Farnesol is a key derivative in the sterol biosynthesis pathway in eukaryotic cells previously identified as a quorum sensing molecule in the human fungal pathogen Candida albicans. Recently, we demonstrated that above threshold concentrations, farnesol is capable of triggering apoptosis in C. albicans. However, the exact mechanism of farnesol cytotoxicity is not fully elucidated. Lipophilic compounds such as farnesol are known to conjugate with glutathione, an antioxidant crucial for cellular detoxification against damaging compounds. Glutathione conjugates act as substrates for ATP-dependent ABC transporters and are extruded from the cell. To that end, this current study was undertaken to validate the hypothesis that farnesol conjugation with intracellular glutathione coupled with Cdr1p-mediated extrusion of glutathione conjugates, results in total glutathione depletion, oxidative stress and ultimately fungal cell death. The combined findings demonstrated a significant decrease in intracellular glutathione levels concomitant with up-regulation of CDR1 and decreased cell viability. However, addition of exogenous reduced glutathione maintained intracellular glutathione levels and enhanced viability. In contrast, farnesol toxicity was decreased in a mutant lacking CDR1, whereas it was increased in a CDR1-overexpressing strain. Further, gene expression studies demonstrated significant up-regulation of the SOD genes, primary enzymes responsible for defense against oxidative stress, with no changes in expression in CDR1. This is the first study describing the involvement of Cdr1p-mediated glutathione efflux as a mechanism preceding the farnesol-induced apoptotic process in C. albicans. Understanding of the mechanisms underlying farnesol-cytotoxicity in C. albicans may lead to the development of this redox-cycling agent as an alternative antifungal agent.法尼醇（farnesol）是真核细胞固醇生物合成通路中的关键衍生物，此前被鉴定为人类真菌病原体白色念珠菌（Candida albicans）的群体感应分子。近期本团队证实，当浓度超过阈值时，法尼醇可诱导白色念珠菌发生细胞凋亡，但法尼醇细胞毒性的确切机制尚未完全阐明。已知脂溶性化合物（如法尼醇）可与谷胱甘肽（glutathione）结合——谷胱甘肽是一种至关重要的抗氧化剂，可帮助细胞抵御有害化合物完成解毒过程。谷胱甘肽结合物可作为ATP依赖型ABC转运蛋白的底物，并被排出细胞外。基于此，本研究旨在验证下述假说：法尼醇与细胞内谷胱甘肽结合，并经Cdr1p介导排出谷胱甘肽结合物，最终会导致总谷胱甘肽耗竭、氧化应激，乃至真菌细胞死亡。联合实验结果显示，细胞内谷胱甘肽水平显著下降，同时伴随CDR1基因的上调以及细胞活力降低。但添加外源性还原型谷胱甘肽可维持细胞内谷胱甘肽水平，并提升细胞活力。与之相反，在缺失CDR1的突变菌株中法尼醇的毒性有所减弱，而在CDR1过表达菌株中其毒性则增强。进一步的基因表达研究表明，负责氧化应激防御的核心酶——超氧化物歧化酶（SOD，superoxide dismutase）基因的表达显著上调，而CDR1的表达无明显变化。本研究首次阐明了Cdr1p介导的谷胱甘肽外排是法尼醇诱导白色念珠菌细胞凋亡过程中的上游机制。阐明法尼醇对白色念珠菌的细胞毒性机制，有望推动这类氧化循环剂开发为新型抗真菌疗法。