Rv0494蛋白在结核分枝杆菌持留中的作用及分子机理

Tuberculosis (TB), which caused by the highly infectious bacterial pathogen Mycobacterium tuberculosis (MTB), remains a major global health threat. The emergence of multi-drug resistant and extensively drug resistant strains of MTB further complicates this problem. MTB is a facultative parasitic bacterium, after being engulfed by macrophages can enter persistent stage. It has been reported that the MTB persister use fatty acids as sole source of nutrition, their growing energy needed to get through the glyoxylate cycle, resulting in the ability of macrophages persistent, Isocitrate lyase (ICL) is the first key enzyme of the glyoxylate cycle. E. coli rely on fatty acid metabolism regulator (FadR) to regulate the metabolism of fatty acids, and the transcription of ICL. Our previous study suggests that Rv0494 protein could regulate the metabolism of fatty acids, and enhance the persistence of mycobacterium within oxidative stress. Rv0494 shows enhanced expression during nutrient starvation was also shown by other researchers. This project intends to design appropriate experiments to further study the mechanism and function of Rv0494 protein in MTB persister, guiding the development of new anti-TB drugs.

耐多药和广泛耐药结核病日趋严重，成为结核病防控的重大挑战。其致病菌结核分枝杆菌是胞内致病菌，被巨噬细胞吞噬后可进入持留状态。该状态是导致结核病长疗程用药、治疗后易复发并产生耐药的主要原因。持留状态结核杆菌仅能以脂肪酸为碳源，通过乙醛酸循环获取能量。ICL是乙醛酸循环的关键酶，大肠杆菌通过FadR调节ICL的表达和脂肪酸代谢。我们前期研究发现结核杆菌Rv0494蛋白类似大肠杆菌FadR的功能，可影响脂肪酸代谢，增强结核杆菌应对氧化压力的能力，提示其与结核杆菌持留密切相关。有证据显示Rv0494蛋白在结核杆菌处于持留状态时表达明显上调。本项目拟采用基因敲除、Western-Blot、RT-PCR等方法，通过研究氧化、强酸及抗生素条件下，Rv0494蛋白对结核杆菌持留菌形成的影响，结合结核杆菌侵染巨噬细胞后，巨噬细胞凋亡及靶基因表达情况，阐明Rv0494蛋白在结核杆菌维持其持留状态中的分子机制。

耐多药和广泛耐药结核病日趋严重，成为结核病防控的重大挑战。其致病菌结核分枝杆菌是胞内致病菌，被巨噬细胞吞噬后可进入持留状态。该状态是导致结核病长疗程用药、治疗后易复发并产生耐药的主要原因。持留状态结核分枝杆菌仅能以脂肪酸为碳源，通过乙醛酸循环获取能量。ICL是乙醛酸循环的关键酶，大肠杆菌通过FadR调节ICL的表达和脂肪酸代谢。本项目采用定位突变、分子杂交、靶基因敲除等实验方法，分别构建了耻垢分枝杆菌和结核分枝杆菌的FadRE.coli同源序列缺失及功能回复突变株。通过模拟极端环境和药敏实验，证明分枝杆菌中FadR蛋白功能的缺失，会影响其持留子的形成。由于FadR可能亦参与其他细菌持留子的形成，我们的研究结果支持FadR可以作为一个针对持留菌的新药物靶标，以改善持留性细菌感染的治疗。