Porins of Mycobacterium tuberculosis

描述(申请人提供)：结核病(TB)是一个主要的全球健康问题，每年导致约200万人死亡。结核病化疗的时间长短、耐多药菌株的传播日益增多，以及目前影响到约三分之一人口的结核分枝杆菌持续感染未能得到治疗，这些都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，这是结核分枝杆菌在体内和/或体外生存所必需的。其中一个目标是利用高通量筛选技术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)极低的渗透性，使分枝杆菌对许多抗生素具有内在耐药性。四种一线结核病药物中有三种被认为是通过充满水的通道蛋白进入结核分枝杆菌的OM。这些孔蛋白是分枝杆菌吸收亲水性溶质的关键蛋白质。我们最近发现了两种结核分枝杆菌孔蛋白，它们补充了耻垢分枝杆菌孔蛋白突变体的通透性缺陷。在脂质双层实验中，两种纯化的重组蛋白都显示了通道活性。我们想要分析孔蛋白介导的OM通透性在体外和体内对结核分枝杆菌的生理作用，并了解有效地将药物转移到结核分枝杆菌的要求。我们将通过筛选牛分枝杆菌转座子文库和耻垢分枝杆菌孔蛋白突变株中结核分枝杆菌的表达文库来鉴定进一步的孔蛋白基因。我们将构建结核分枝杆菌的孔蛋白突变体，并通过转运实验检测相应的孔蛋白对OM通透性的作用以及对体外和小鼠生长的影响。通过对结核分枝杆菌孔蛋白突变体的敏感性和转运实验，将分析孔蛋白在结核分枝杆菌对抗生素和当前结核病药物敏感性中的作用。将从结核分枝杆菌中提纯主要的孔蛋白，并在脂双层和脂质体溶胀实验中分析其通道特性。该蛋白将在大肠杆菌中过度表达，折叠和纯化，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，而且对我们理解结核分枝杆菌的营养吸收也是有根本意义的。

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.