Porins of Mycobacterium tuberculosis

结核分枝杆菌孔蛋白

基本信息

批准号：
7559647
负责人：
MICHAEL NIEDERWEIS
金额：
$ 33.83万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-02-01 至 2010-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7559647
关键词：
Affect Amino Acids Antibiotics Antitubercular Agents Bacteriophages Cell Membrane Permeability Cells Cessation of life Charge Complement Defect Diffusion Doctor of Philosophy Drug Delivery Systems Drug Transport Enzyme-Linked Immunosorbent Assay Escherichia coli Exhibits Expression Library Failure Genes Genus Mycobacterium Goals Growth Health Human In Vitro Infection Lead Length Libraries Lipid Bilayers Liposomes Mediating Membrane Molecular Monoclonal Antibodies Multi-Drug Resistance Mus Mycobacterium smegmatis Mycobacterium tuberculosis Nutrient Permeability Pharmaceutical Preparations Physiological Population Predisposition Property Proteins Recombinant Proteins Recombinants Research Personnel Resistance Role Screening procedure Structure Survival Analysis Swelling Technology Tuberculosis Virulence Water Western Blotting chemotherapy design high throughput screening in vivo interest mutant mycobacterial porin programs research study resistant strain solute sugar tuberculosis drugs tuberculosis treatment uptake

项目摘要

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.

描述（由申请人提供）：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。结核病化疗的时间较长、多重耐药菌株的日益传播以及目前治疗影响约三分之一人口的结核分枝杆菌持续感染的失败，都加大了全世界寻找新抗结核药物的努力。新药的研究重点是蛋白质，这是结核分枝杆菌在体内和/或体外存活所必需的。目标之一是利用高通量筛选技术鉴定新的先导化合物。然而，结核病化疗的主要困难不是寻找新的药物靶点，而是异常的分枝杆菌外膜（OM）的极低渗透性使得分枝杆菌对许多抗生素具有内在耐药性。据推测，四分之三的一线结核病药物可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质。我们最近发现了结核分枝杆菌的两种孔蛋白，可以补充耻垢分枝杆菌孔蛋白突变体的通透性缺陷。两种纯化的重组蛋白在脂质双层实验中均显示出通道活性。我们想要在体外和体内分析孔蛋白介导的 OM 渗透性对结核分枝杆菌的生理作用，并了解有效药物转运到结核分枝杆菌的要求。我们将通过筛选牛分枝杆菌BCG转座子文库和耻垢分枝杆菌孔蛋白突变体中的结核分枝杆菌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，并通过转运实验以及体外和小鼠体内的生长来检查相应孔蛋白对 OM 通透性的功能。将通过敏感性和结核分枝杆菌孔蛋白突变体的转运实验来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纯化主要孔蛋白，以分析其在脂质双层和脂质体溶胀实验中的通道特性。该蛋白质将在大肠杆菌中过度表达、折叠和纯化以确定其晶体结构。这些研究不仅对于设计新的结核病药物至关重要，而且对于我们了解结核分枝杆菌的营养吸收也具有根本意义。

项目成果

期刊论文数量（16）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Mycobacterial outer membranes: in search of proteins.

DOI：
10.1016/j.tim.2009.12.005
发表时间：
2010-03
期刊：
Trends in microbiology
影响因子：
15.9
作者：
Niederweis M;Danilchanka O;Huff J;Hoffmann C;Engelhardt H
通讯作者：
Engelhardt H

Construction of unmarked deletion mutants in mycobacteria.

分枝杆菌中未标记缺失突变体的构建。

DOI：
10.1007/978-1-59745-207-6_19
发表时间：
2009
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
0
作者：
Song,Houhui;Wolschendorf,Frank;Niederweis,Michael
通讯作者：
Niederweis,Michael

Physiology of mycobacteria.

DOI：
10.1016/s0065-2911(09)05502-7
发表时间：
2009
期刊：
ADVANCES IN MICROBIAL PHYSIOLOGY
影响因子：
0
作者：
Cook, Gregory M.;Berney, Michael;Gebhard, Susanne;Heinemann, Matthias;Cox, Robert A.;Danilchanka, Olga;Niederweis, Michael
通讯作者：
Niederweis, Michael

Porins are required for uptake of phosphates by Mycobacterium smegmatis.

耻垢分枝杆菌吸收磷酸盐需要孔蛋白。