Translation Regulation Promotes Stress Adaptation in Candida glabrata

Candida glabrata is an important human fungal pathogen leading cause of non-albicans Candida infections. C. glabrata exhibits resistance to key antifungal drugs, rapidly replicates and divides in host macrophages and withstands highly stressful host conditions. This study explores the molecular mechanisms underlying stress adaptations in C. glabrata that contribute to its pathogenicity. Our findings revealed that C. glabrata survives oxidative stress and amino acid starvation more effectively than S. cerevisiae, C. albicans, and C. auris. We noted that Gcn2 kinase and Gcn4 play critical roles in this adaptation as Gcn2 phosphorylates eIF2a and downregulates the global protein translation, activating GCN4. RNA sequencing of WT and gcn4 mutant revealed that GCN4 activation during stress orchestrates the expression of stress-responsive genes vital for survival during amino acid starvation and oxidative stress. Ultimately assisting in the stress adaptative transcriptome. Thus, this study highlights the critical role of the Gcn2–Gcn4 pathway in stress adaptation in C. glabrata. Overall design: The WT and gcn4 mutant at log phase O.D. in Simple Dextrose (SD) minimal media were treated with 5 mM 3AT (3-amino triazole) that provided histidine starvation and ultimately amino acid starvation and 10 mM H2O2 (Hydrogen peroxide) as a ROS agent generating oxidative stress. Both treatments were given for 30 minutes keeping the untreated control with three individual replicates. The cells were pelleted, and total RNA was extracted from the untreated and treated samples for both stress and biological replicates. Samples were then sent for transcriptome profiling.

光滑念珠菌（Candida glabrata）是一类重要的人类真菌病原体，也是非白假丝酵母菌感染的首要致病原。该菌对关键抗真菌药物具有耐药性，可在宿主巨噬细胞内快速增殖与分裂，并耐受宿主内的高应激微环境。本研究旨在解析光滑念珠菌应激适应的分子机制，该机制与其致病性密切相关。

研究结果显示，相较于酿酒酵母（S. cerevisiae）、白色念珠菌（C. albicans）及耳念珠菌（C. auris），光滑念珠菌在氧化应激与氨基酸饥饿条件下的存活能力显著更强。我们发现，Gcn2激酶与Gcn4在该应激适应过程中发挥核心调控作用：Gcn2可磷酸化真核翻译起始因子2α（eIF2α），下调全局蛋白质翻译过程，进而激活GCN4。对野生型（Wild Type, WT）与gcn4突变体的RNA测序结果表明，应激状态下GCN4的激活可调控参与氨基酸饥饿与氧化应激存活的关键应激响应基因的表达，最终塑造适应性应激转录组。综上，本研究揭示了Gcn2–Gcn4通路在光滑念珠菌应激适应中的关键作用。

整体实验设计：将处于对数生长期、光密度（Optical Density, O.D.）一致的野生型与gcn4突变体菌株接种于简单葡萄糖（SD）基本培养基中，分别采用5 mM 3-氨基三唑（3-amino triazole, 3AT）处理以诱导组氨酸饥饿，进而引发全面的氨基酸饥饿；同时采用10 mM过氧化氢（H₂O₂）作为活性氧（Reactive Oxygen Species, ROS）诱导剂，构建氧化应激模型。两种应激处理均持续30分钟，同时设置未处理的对照组，每组均设置3个生物学重复。收集细胞沉淀后，从未处理样本、应激处理样本及各生物学重复样本中提取总RNA，随后将所有样本送往开展转录组测序分析。