放线菌胆固醇降解中TetR家族转录调控因子的作用机理

Bacterial catabolism of cholesterol plays an important role in Mycobacterium tuberculosis(Mtb) infection and intracellular survival. The mechanism about the uptake and utilization of cholesterol by Mtb within the host cell is still poorly understood. TetR type transcriptional regulators are a group of proteins that are involved in a diverse array of cellular functions. In our previous work, a draft sequence of the genome of Gordonia neofelifaecis, a cholesterol-degrading actinomycete was analyzed, the results showed that 9 putative TetR-type transcriptional regulator genes lie within a gene cluster encoding cholesterol catabolism, different from those of Mycobacterium tuberculosis in which 2 TetR-type transcriptional regulators would control 98 genes involved in cholesterol utilization. The objective of this application is to determine the mechanisms regulating the cholesterol catabolism in G. neofelifaecis. We propose herein that the cholesterol-degrading genes, which are responsible for a specific degrading reaction or catabolism of a kind of steroid, are controlled precisely by different TetR-type transcriptional regulators. And one the transcriptional regulators may responsible for one degrading reaction or the degradation of a kind of steroid with different structure, respectively. In present project, we will utilise both transcriptomic and genetic strategies to dissect the role of these candidate regulators in the emergence of steroids, and determine the effector molecule,downstream genes of them and helps to assign genes to specific metabolic pathways. Additionally, the gene regulatory networks will also be investigated. Those genes that involve in cholesterol degradation would represent potential targets for antibacterial drug interventions.

结核分枝杆菌的胆固醇分解代谢对其侵染和胞内存活起着十分重要的作用。但有关微生物对胆固醇的降解及其调控机理，还知之甚少。本课题拟以放线菌类的胆固醇降解菌-戈登氏菌为研究对象，揭示胆固醇分解代谢调控的分子机理。在前期工作中，我们对戈登氏菌进行了全基因组测序，发现胆固醇降解基因簇有多达9个可能的TetR家族转录调控因子编码基因，分布于各操纵子中；最近有研究报道结核分枝杆菌胆固醇降解受到2个TetR家族成员调控。基于此，本项目提出假想：各个胆固醇降解操纵子通过TetR转录因子调控，分别负责某一种类固醇的降解或某一特定反应，从而实现整个基因簇的协调表达。本项目拟从获得的9个TetR基因着手，采用基因敲除、RNA-Seq、DNA电泳阻滞实验等技术，筛选出参与胆固醇降解代谢的新调控基因，明确其诱导物、下游基因及调控通路，阐明胆固醇降解基因协同表达的机理，进而为结核杆菌治疗提供新的药物靶标。

本研究首先利用大肠杆菌-分枝杆菌穿梭质粒(pNIT, pTACT-1、 pJAK2A)对3株胆固醇降解菌（包括戈登氏菌、耻垢分枝杆菌、Mycobacterium vaccae）的电转化进行了研究，获得了优化的转化条件；利用RNA-seq测序技术分析了戈登氏菌、耻垢分枝杆菌2株胆固醇降解菌的转录组。以位于胆固醇降解基因簇中、差异表达的、TetR调控因子编码基因MSMEG_6032, MSMEG_5040为研究对象，开展了基因的克隆与异源表达研究，并构建了基因敲除质粒，对耻垢分枝杆菌进行了遗传转化，筛选获得了多个转化子。对存在于戈登氏菌基因组中5个可能的3-甾酮-△1-脱氢酶基因（kstD1 to kstD5）进行了克隆，并在大肠杆菌中实现了异源表达，经分离纯化后，分析其对多种固醇化合物的催化活性。目前，项目仍在不断的开展之中。