Data_Sheet_1_Understanding Metabolic Remodeling in Mycobacterium smegmatis to Overcome Energy Exigency and Reductive Stress Under Energy-Compromised State.pdf

Mycobacteria such as Mycobacterium tuberculosis, the causative agent of tuberculosis that annually kills several million people worldwide, and Mycobacterium smegmatis, the non-pathogenic fast-growing mycobacteria, require oxidative phosphorylation to meet their energy requirements. We have previously shown that deletion of one of the two copies of atpD gene that codes for the ATP synthase β-subunit establishes an energy-compromised state in M. smegmatis. Here we report that upon such deletion, a major routing of electron flux occurs through the less energy-efficient complexes of its respiratory chain. ΔatpD bacterium also shows an increased reduced state which is further confirmed by the overexpression of WhiB3, a major redox sensor. We show a substantial modulation of the biosynthesis of cell wall associated lipids and triacylglycerol (TAG). An accumulation of TAG-containing lipid bodies is further confirmed by using 14C oleate incorporation. Interestingly, the mutant also shows an overexpression of TAG-degrading lipase genes, and the intracellular lipolytic enzymes mediate TAG hydrolysis for their utilization as energy source. We believe that our in vitro energy-depleted model will allow us to explore the critical link between energy metabolism, redox homeostasis, and lipid biosynthesis during ATP-depleted state, which will enhance our understanding of the bacterial adaptation, and will allow us to identify novel drug targets to counter mycobacterial infections.

诸如每年在全球造成数百万人口死亡的结核病致病菌结核分枝杆菌（Mycobacterium tuberculosis），以及非致病性快速生长分枝杆菌耻垢分枝杆菌（Mycobacterium smegmatis）等分枝杆菌，均依赖氧化磷酸化满足自身能量需求。我们此前的研究证实，编码ATP合酶β亚基（ATP synthase β-subunit）的atpD基因的两个拷贝之一发生缺失后，会使耻垢分枝杆菌陷入能量受损状态。本研究报道，发生此类缺失后，其呼吸链中的电子流会主要通过能量利用效率更低的复合物进行传递。ΔatpD菌株还呈现出还原态升高的表型，这一点可通过主要氧化还原传感器WhiB3的过表达得到进一步验证。我们发现，细胞壁相关脂质以及三酰甘油（triacylglycerol, TAG）的生物合成发生了显著调控。通过14C标记油酸掺入实验，进一步证实了含TAG的脂滴的积累。值得注意的是，该突变体同时表现出TAG降解脂肪酶基因的过表达，胞内脂解酶可介导TAG水解，将其作为能量来源加以利用。我们认为，本研究构建的体外能量耗竭模型，将有助于探索ATP耗竭状态下能量代谢、氧化还原稳态与脂质生物合成之间的关键关联，从而加深我们对细菌适应性机制的理解，并为对抗分枝杆菌感染的新型药物靶点筛选提供新思路。