Porins of Mycobacterium tuberculosis

描述（由申请人提供）：结核病（TB）是全球主要的健康问题，每年造成约200万人死亡。结核病化疗时间的延长、耐多药菌株的日益扩散以及目前无法治疗影响约三分之一人口的结核分枝杆菌持续性感染，这些都加强了世界范围内寻找新的抗结核药物的努力。寻找新药的重点是蛋白质，这些蛋白质是结核分枝杆菌在体内和/或体外存活所必需的。一个目标是利用高通量筛选技术鉴定新的先导化合物。然而，结核化疗的主要困难不是寻找新的药物靶点，而是罕见的分枝杆菌外膜（OM）的极低通透性使得分枝杆菌对许多抗生素具有内在耐药性。假定四种一线结核病药物中有三种通过充满水的通道蛋白穿过结核分枝杆菌的OM。这些孔蛋白是分枝杆菌吸收亲水性溶质的关键蛋白。我们最近发现结核分枝杆菌的两个孔蛋白，它们补充了耻垢分枝杆菌的一个孔蛋白突变体的渗透性缺陷。两种纯化的重组蛋白在脂质双分子层实验中均表现出通道活性。我们希望在体外和体内分析孔蛋白介导的OM通透性对结核分枝杆菌的生理作用，并了解有效的药物转运到结核分枝杆菌中的要求。我们将通过筛选牛分枝杆菌卡介苗的转座子文库和结核分枝杆菌在耻垢分枝杆菌的孔蛋白突变体中的表达文库来进一步鉴定孔蛋白基因。我们将构建结核分枝杆菌的孔蛋白突变体，并通过转运实验和体外及小鼠生长研究相应的孔蛋白对OM通透性的功能。通过对结核分枝杆菌的敏感性和结核分枝杆菌孔蛋白突变体的转运实验，我们将分析孔蛋白在结核分枝杆菌对抗生素和现有结核病药物的敏感性中的作用。从结核分枝杆菌中纯化主要的孔蛋白，分析其在脂质双分子层和脂质体肿胀实验中的通道特性。该蛋白将在大肠杆菌中过度表达，折叠并纯化以确定其晶体结构。这些研究不仅对设计新的结核病药物至关重要，而且对我们了解结核分枝杆菌的营养吸收也具有重要意义。

DESCRIPTION (provided by applicant): Tuberculosis (TB) is a major global health problem causing about two million deaths per year. The length of TB chemotherapy, the increasing spread of multi-drug resistant strains and the current failure to treat persistent infections with Mycobacterium tuberculosis affecting approximately one-third of the human population, have intensified worldwide efforts to find new antitubercular drugs. The search for new drugs is focused on proteins, which are required for survival of M. tuberculosis in vivo and/or in vitro. One goal is to identify new lead compounds using high-throughput screening technologies. However, the major difficulty in TB chemotherapy is not finding new drug targets, but rather the extremely low permeability of the unusual mycobacterial outer membrane (OM) rendering mycobacteria intrinsically resistant against many antibiotics. Three out of four first-line TB drugs are assumed to cross the OM of M. tuberculosis by water-filled channel proteins. These porins are the key proteins for uptake of hydrophilic solutes in mycobacteria. We recently discovered two porins of M. tuberculosis that complement the permeability defects of a porin mutant of M. smegmatis. Both purified recombinant proteins showed channel activity in lipid bilayer experiments. We want to analyze the physiological role of the porin-mediated OM permeability for M. tuberculosis in vitro and in vivo and to understand the requirements for efficient drug transport into M. tuberculosis. We will identify further porin genes by screening a transposon library of M. bovis BCG and an expression library of M. tuberculosis in a porin mutant of M. smegmatis. We will construct porin mutants of M. tuberculosis and examine the function of the corresponding porins for OM permeability by transport experiments and for growth in vitro and in mice. The role of porins for susceptibility of M. tuberculosis to antibiotics and current TB drugs will be analyzed both by sensitivity and by transport experiments with M. tuberculosis porin mutants. The main porin will be purified from M. tuberculosis to analyze its channel properties in lipid bilayer and liposome swelling experiments. The protein will be over expressed in E. coli, folded and purified to determine its crystal structure. These studies will not only be essential for the design of new TB drugs, but will also be of fundamental interest for our understanding of nutrient uptake by M. tuberculosis.