School of Chemistry and Chemical Engineering, Shihezi University, Xinjiang, China.
Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Biotechnol Appl Biochem. 2022 Jun;69(3):1209-1216. doi: 10.1002/bab.2197. Epub 2021 May 27.
Tuberculosis, caused by mycobacteria, continues to pose a substantial public health threat. Mycobacteria typically use cholesterol from the membranes of host macrophages as a carbon and energy source. Most genes that control cholesterol degradation are regulated by KstR, which is highly conserved in Mycobacterium tuberculosis and Mycobacterium smegmatis. Through bioinformatic analysis, we found a typical global nitrogen regulator (GlnR)-binding motif (CCGAC-AACAGT-GACAC) in the promoter region of kstR of M. smegmatis, and we determined its binding activity in vitro using electrophoretic mobility shift assays. Using RT-qPCR, we found that nine genes involved in side-chain or sterol-ring oxidation were upregulated in a ΔglnR M. smegmatis strain compared to the WT strain and glnR-complemented strains under nitrogen limitation. ATP assays in macrophages revealed that coordinated GlnR-KstR regulation significantly reduced the viability of M. smegmatis in macrophages. Thus, we found that various genes involved in cholesterol catabolism are regulated by GlnR via KstR in response to environmental nitrogen, and that they further affect the invasive ability of M. smegmatis. These findings revealed a novel regulatory mechanism of cholesterol catabolism, which may be useful in the development of new strategies for controlling tuberculosis.
结核病是由分枝杆菌引起的，仍然对公共卫生构成重大威胁。分枝杆菌通常将宿主巨噬细胞膜中的胆固醇作为碳源和能源。控制胆固醇降解的大多数基因都受到 KstR 的调节，KstR 在结核分枝杆菌和耻垢分枝杆菌中高度保守。通过生物信息学分析，我们在耻垢分枝杆菌 kstR 的启动子区域发现了一个典型的全局氮调节因子 (GlnR) 结合基序 (CCGAC-AACAGT-GACAC)，并通过电泳迁移率变动分析在体外确定了其结合活性。使用 RT-qPCR，我们发现与野生型菌株和氮限制下的 glnR 互补菌株相比，氮限制下的ΔglnR 耻垢分枝杆菌菌株中，有九个参与侧链或甾环氧化的基因上调。在巨噬细胞中的 ATP 测定表明，GlnR-KstR 协调调节可显著降低分枝杆菌在巨噬细胞中的活力。因此，我们发现胆固醇代谢的各种基因通过 KstR 响应环境氮被 GlnR 调节，并且它们进一步影响耻垢分枝杆菌的侵袭能力。这些发现揭示了胆固醇代谢的一种新的调控机制，这可能有助于开发控制结核病的新策略。
