The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong, China; Institute of Mycology, Jinan University, Guangzhou, Guangdong, China.
New Drug Research and Development Center, School of Pharmacy, Second Military Medical University, Shanghai, China.
Int J Med Microbiol. 2019 Sep;309(6):151330. doi: 10.1016/j.ijmm.2019.151330. Epub 2019 Aug 17.
Alcohol dehydrogenase I is encoded by ADH1 in Candida albicans, and is one of the key enzymes in fungal metabolism by which it catalyzes the conversion from acetaldehyde to ethanol. The role of the associated protein Adh1p, encoded by ADH1 in fungal pathogenicity has not been thoroughly studied despite its near ubiquity in the fungal kingdom. Using C. albicans as a model, this study proposes to determine the possible pathogenic roles for ADH1 and its possible underlying mechanisms.
The SAT1 flipper strategy was used to construct the ADH1 deletion mutant. Growth curves and spot assay were used to compare growth and cell viability of the mutant to wild type C. albicans. Three host model systems (infected mice, C. elegans, and G. mellonella) were used to investigate the effects of ADH1 deletion in vivo on C. albicans pathogenicity. Then, adhesion, hyphal formation, biofilm formation, cell surface hydrophobicity (CSH) and RT-qPCR were performed to investigate the effects of ADH1 deletion in vitro on C. albicans virulence. Finally, Xfe 96 seahorse assay, ROS level, mitochondrial membrane potential, and intracellular ATP content were used to determine the effects of ADH1 deletion on bioenergetics.
ADH1 deletion has no effects on the growth and cell viability of C. albicans, but significantly prolongs survival time in each of the three host models, decreases fungal burden in kidney and liver, and lessens pathological tissue damage (P < 0.05). In addition, ADH1 deletion significantly increases CSH and reduces C. albicans virulence in terms of adhesion, hyphal formation and biofilm formation in accord with the downregulation of virulence-related genes such as ALS1, ALS3, HWP1, and CSH1 (P < 0.05). For bioenergetics, ADH1 deletion has no obvious effect on glycolysis, but a lack of ADH1 significantly increases ROS levels and decreases mitochondrial membrane potential and intracellular ATP content even through the mitochondrial oxygen consumption rate and NADH/NAD ratio are elevated (P < 0.05).
Our results suggest that the fermentative enzyme ADH1 is required for the pathogenicity of C. albicans under one of the presumed mechanisms viaits effects on oxidative phosphorylation activities in mitochondria.
在白色念珠菌中，乙醇脱氢酶 I 由 ADH1 编码，是真菌代谢中的关键酶之一，可催化乙醛转化为乙醇。尽管 ADH1 在真菌界中普遍存在，但与其相关的蛋白 Adh1p 在真菌致病性中的作用尚未得到彻底研究。本研究以白色念珠菌为模型，旨在确定 ADH1 及其可能的潜在机制的可能致病作用。
使用 SAT1 翻转策略构建 ADH1 缺失突变体。生长曲线和点试验用于比较突变体和野生型白色念珠菌的生长和细胞活力。使用三种宿主模型系统（感染小鼠、秀丽隐杆线虫和地中海实蝇）研究 ADH1 缺失对白色念珠菌体内致病性的影响。然后，进行粘附、菌丝形成、生物膜形成、细胞表面疏水性 (CSH) 和 RT-qPCR，以研究 ADH1 缺失对白色念珠菌毒力的影响。最后，使用 Xfe 96 Seahorse 测定法、ROS 水平、线粒体膜电位和细胞内 ATP 含量，以确定 ADH1 缺失对生物能量的影响。
ADH1 缺失对白色念珠菌的生长和细胞活力没有影响，但在三种宿主模型中的每一种中都显著延长了存活时间，降低了肾脏和肝脏中的真菌负荷，并减轻了病理组织损伤（P<0.05）。此外，ADH1 缺失显著增加了 CSH，并降低了白色念珠菌的粘附、菌丝形成和生物膜形成的毒力，与 ALS1、ALS3、HWP1 和 CSH1 等毒力相关基因的下调一致（P<0.05）。对于生物能量，ADH1 缺失对糖酵解没有明显影响，但缺乏 ADH1 会显著增加 ROS 水平，并降低线粒体膜电位和细胞内 ATP 含量，即使线粒体耗氧量和 NADH/NAD 比升高（P<0.05）。
我们的结果表明，发酵酶 ADH1 通过其对线粒体氧化磷酸化活性的影响，是白色念珠菌致病性所必需的，这是一种假定的机制之一。
