Porins of Mycobacterium tuberculosis

描述（由申请人提供）：结核病（TB）是一个主要的全球性健康问题，每年造成约200万人死亡。结核病化疗的持续时间、多药耐药菌株的日益蔓延以及目前未能治疗影响约三分之一人口的结核分枝杆菌持续感染，已加强了全世界寻找新的抗结核药物的努力。寻找新药的重点是蛋白质，这是M生存所必需的。体内和/或体外的结核病。一个目标是使用高通量筛选技术来识别新的先导化合物。然而，结核病化疗的主要困难不是寻找新的药物靶点，而是不寻常的分枝杆菌外膜（OM）的极低渗透性，使分枝杆菌对许多抗生素具有固有的耐药性。据推测，四分之三的一线结核病药物可以通过M的OM。水通道蛋白引起的结核病。这些孔蛋白是分枝杆菌摄取亲水性溶质的关键蛋白。我们最近发现了M.结核分枝杆菌的孔蛋白突变体的渗透性缺陷的补充。恶臭这两种纯化的重组蛋白在脂质双层实验中显示出通道活性。我们想分析孔蛋白介导的OM渗透性对M.结核病在体外和体内，并了解有效的药物转运到M。结核我们将通过筛选M. bovis BCG和M.结核分枝杆菌的孔蛋白突变体。恶臭我们将构建M.结核病和检查相应的孔蛋白的功能OM渗透通过运输实验和生长在体外和小鼠。孔蛋白在M.结核病对抗生素和当前结核病药物的敏感性和用M.结核孔蛋白突变体。主要的孔蛋白将从M.在脂质双层和脂质体溶胀实验中分析其通道特性。该蛋白在E.大肠杆菌，折叠和纯化，以确定其晶体结构。这些研究不仅对新的结核病药物的设计至关重要，而且对我们理解M.结核

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.