Candida species causes superficial and life-threatening systemic infections and are difficult to treat due to the resistance of these organism to various clinically used drugs. Protolichesterinic acid is a well-known lichen compound. Although the antibacterial activity of protolichesterinic acid has been reported earlier, the antifungal property and its mechanism of action are still largely unidentified. The goal of the present investigation is to explore the anticandidal activity and mechanism of action of protolichesterinic acid, especially against Candida tropicalis. The Minimum Inhibitory Concentration (MIC) value was established through microdilution techniques against four Candida species and out of four species tested, C. tropicalis showed a significant effect (MIC: 2 μg/ml). In the morphological interference assay, we observed the enhanced inhibition of hyphae when the cells were treated with protolichesterinic acid. Time-kill assay demonstrated that the maximum rate of killing was recorded between 2 and 6 h. C. tropicalis exposed to protolichesterinic acid exhibited an increased ROS production, which is one of the key factors of fungal death. The rise in ROS was due to the dysfunction of mitochondria caused by protolichesterinic acid. We confirmed that protolichesterinic acid-induced dysfunction of mitochondria in C. tropicalis. The damage of cell membrane due to protolichesterinic acid treatment was confirmed by the influx of propidium iodide and was further confirmed by the release of potassium ions. The treatment of protolichesterinic acid also triggered calcium ion signaling. Moreover, it commenced apoptosis which is clearly evidenced by Annexin V and propidium iodide staining. Interestingly protolichesterinic acid recorded excellent immunomodulatory property when tested against lymphocytes. Finally protolichesterinic acid showed low toxicity toward a normal human cell line Foreskin (FS) normal fibroblast. In in vivo test, protolichesterinic acid significantly enhanced the survival of C. tropicalis infected Caenorhabditis elegans. This investigation proposes that the protolichesterinic acid induces apoptosis in C. tropicalis via the enhanced accumulation of intracellular ROS and mitochondrial damage, which leads fungal cell death via apoptosis. Our work revealed a new key aspect of mechanisms of action of protolichesterinic acid in Candida species. This article is the first study on the antifungal and mechanism of action of protolichesterinic acid in Candida species.

中文翻译：

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来自地衣松萝的原脂城素酸的抗真菌机制在于细胞内活性氧的积累和线粒体介导的细胞凋亡导致的热带念珠菌死亡。

念珠菌引起浅表和危及生命的全身感染，由于这些生物体对各种临床使用的药物具有抗性，因此难以治疗。原生鸟酸是一种众所周知的地衣化合物。尽管原扁桃体酸的抗菌活性已有较早的报道，但其抗真菌特性及其作用机理仍未明确。本研究的目的是探究原扁桃体酸，尤其是针对热带假丝酵母的抗candidal活性和作用机理。通过微稀释技术确定了针对四种念珠菌的最低抑菌浓度（MIC）值，并且在所测试的四种菌种中，热带假丝酵母显示出显着效果（MIC：2μg/ ml）。在形态干扰分析中，我们观察到当用原脂切力酸处理细胞时，增强了对菌丝的抑制作用。时间杀灭试验证明最大杀灭率记录在2至6小时之间。暴露于原扁桃酸的热带假丝酵母表现出增加的ROS产生，这是真菌死亡的关键因素之一。ROS的升高是由于原肠酸引起的线粒体功能障碍。我们证实原扁桃酸诱导的热带线虫线粒体功能障碍。碘化丙啶的涌入证实了原扁桃素酸处理对细胞膜的损害，钾离子的释放进一步证实了这种破坏。降钙素原酸的治疗也触发了钙离子信号传导。而且，它开始凋亡，膜联蛋白V和碘化丙啶染色清楚地证明了这一点。有趣的是，当针对淋巴细胞进行测试时，原扁桃体酸记录了出色的免疫调节特性。最终，原扁桃酸对正常人细胞系包皮（FS）正常成纤维细胞显示出低毒性。在体内试验中，原扁桃体酸显着提高了感染热带线虫的秀丽隐杆线虫的存活。这项研究表明，原头孢菌素酸通过增强细胞内ROS的积累和线粒体损伤诱导热带梭菌的凋亡，从而通过凋亡导致真菌细胞死亡。我们的工作揭示了念珠菌原酸在念珠菌物种中的作用机理的一个新的关键方面。