Department of Laboratory Medicine, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China.
Department of Clinical Laboratory, Obstetrics and Gynaecology Hospital of Fudan University, Shanghai, China.
Int J Antimicrob Agents. 2023 Jul;62(1):106819. doi: 10.1016/j.ijantimicag.2023.106819. Epub 2023 Apr 16.
Invasive candidiasis is the most common and serious fungal disease worldwide, and the development of antifungal drug resistance in Candida spp. is an emerging problem. Miltefosine, approved as an orphan drug for the therapy of invasive candida infections by the US Food and Drug Administration, has broad-spectrum antifungal activity, but its mechanism of action is unclear. This study evaluated the antifungal drug susceptibility of azole-resistant Candida spp. isolates and found that miltefosine showed good activity, with a geometric mean value of 2 µg/mL. Miltefosine was found to increase production of intracellular reactive oxygen species (ROS) and induce apoptosis in Candida albicans. RNA sequencing (RNA-Seq) analysis and iTRAQ-labelling-based quantitative proteomic mass spectrometry analysis were undertaken. Aif1 and the oxidative stress pathway involved in miltefosine-mediated apoptosis were identified using global transcriptomic and proteomic combined screening. Miltefosine increased mRNA and protein expressions of Aif1. The localization of Aif1 was examined using confocal microscopy, and the GFP-Aif1 fusion protein was found to be translocated from the mitochondria to the nucleus when sensing miltefosine. Next, the pex8 Δ/Δ strain was constructed, and the minimum inhibitory concentration of miltefosine was found to decrease four-fold (from 2 to 0.5 µg/mL) and the intracellular ROS increased significantly after knocking out the PEX8 gene. Moreover, miltefosine was found to trigger Hog1 phosphorylation. These findings indicate that Aif1 activation and the Pex8-mediated oxidative stress pathway are the mechanisms of action of miltefosine on C. albicans. The results help to aid understanding of the mechanisms by which miltefosine acts on fungi.
侵袭性念珠菌病是全球最常见和最严重的真菌感染，念珠菌属对抗真菌药物耐药性的发展是一个新出现的问题。米替福新已被美国食品和药物管理局批准为治疗侵袭性念珠菌感染的孤儿药，具有广谱抗真菌活性，但作用机制尚不清楚。本研究评估了唑类耐药念珠菌属分离株的抗真菌药物敏感性，发现米替福新具有良好的活性，几何平均浓度为 2μg/mL。研究发现米替福新增加了细胞内活性氧（ROS）的产生，并诱导白色念珠菌凋亡。进行了 RNA 测序（RNA-Seq）分析和 iTRAQ 标记定量蛋白质组学质谱分析。使用全转录组和蛋白质组联合筛选鉴定了 Aif1 和与米替福新介导的凋亡相关的氧化应激途径。米替福新增加了 Aif1 的 mRNA 和蛋白表达。使用共聚焦显微镜检查了 Aif1 的定位，发现 GFP-Aif1 融合蛋白在感知米替福新时从线粒体转移到细胞核。接下来构建了 pex8Δ/Δ 菌株，发现敲除 PEX8 基因后米替福新的最小抑菌浓度降低了四倍（从 2μg/mL 降至 0.5μg/mL），细胞内 ROS 显著增加。此外，米替福新被发现触发 Hog1 磷酸化。这些发现表明 Aif1 激活和 Pex8 介导的氧化应激途径是米替福新作用于白色念珠菌的作用机制。研究结果有助于了解米替福新对真菌作用的机制。
