The necrosis-inducing toxin of Mycobacterium tuberculosis

结核分枝杆菌的坏死诱导毒素

• 批准号: 9178625
• 负责人: MICHAEL NIEDERWEIS
• 金额: $ 53.42万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9178625
• 关键词: Antitoxins; Bacterial Infections; Bacterial Toxins; Binding; Binding Sites; Biological Assay; C-terminal; Cell Death; Cells; Cessation of life; Complex; Containment; Crystallization; Cytosol; ELF3 gene; Escherichia coli; Event; Glycoside Hydrolases; Human; Immunity; Infection; Kinetics; Link; Membrane; Membrane Proteins; Molecular; Mutagenesis; Mycobacterium tuberculosis; NAD+ Nucleosidase; Names; Necrosis; Operon; Pathogenesis; Pathway interactions; Peptide Hydrolases; Play; Protein Secretion; Proteins; Role; Structure; System; Toxic effect; Toxin; Tuberculosis; Virulence; Virulence Factors; adaptive immune response; analog; cytotoxicity; enzyme activity; experimental study; genetic selection; global health; killings; macrophage; member; mutant; novel; pathogen; public health relevance

 DESCRIPTION (provided by applicant): Tuberculosis (TB) is one of the most important and widespread bacterial diseases and a global health threat. Survival within infected macrophages is a key feature of Mycobacterium tuberculosis (Mtb) pathogenesis and is crucial to a persistent infection in the human host. The lack of any protein with sequence similarities to bacterial toxins and the apparent absence of a protein toxin with profound effects on host cells led to the widely accepted assumption that Mtb does not have these classical virulence factors. However, this paradigm was challenged by our discovery of the outer membrane protein CpnT which is utilized by Mtb to secrete a toxic C- terminal domain. This toxin is required by Mtb to replicate i macrophages, is the major cytotoxicity factor of Mtb in macrophages and causes necrotic cell death of host cells. Hence, we named this protein Tuberculosis Necrotizing Toxin (TNT). Killing of infected macrophages by necrosis is a well-known, but poorly understood virulence mechanism of Mtb to escape phagosomal containment and the adaptive immune response. Recently, we discovered that TNT is a novel NAD+-glycohydrolase which gains access to the cytosol of infected macrophages in an ESX-1 dependent manner. Mtb protects itself from CpnT/TNT toxicity by producing an antitoxin which we named Immunity Factor of TNT (IFT). IFT inactivates the enzyme activity of TNT upon binding. The discovery of IFT enabled us to produce TNT protein in mg quantities and, subsequently, to solve the crystal structure of the TNT-IFT complex and characterize the purified TNT protein enzymatically. We have identified catalytically inactive TNT mutants, which we will use to define the role of this Mtb toxin in the molecular pathway ultimately leading to necrosis of infected macrophages. Thus, we are now able to examine a novel molecular mechanism of paramount importance for the interaction of Mtb with host cells. These findings will also be important for more than 200 bacterial and fungal pathogens with TNT homologs and for understanding the molecular consequences of NAD+ depletion in host cells.

 描述（申请人提供）：结核病（TB）是最重要和最广泛的细菌性疾病之一，是全球性的健康威胁。在感染的巨噬细胞内存活是结核分枝杆菌（Mtb）发病机制的关键特征，并且对于人类宿主中的持续感染至关重要。缺乏任何与细菌毒素序列相似的蛋白质 并且明显缺乏对宿主细胞具有深远影响的蛋白质毒素导致了广泛接受的假设，即Mtb不具有这些经典的毒力因子。然而，我们发现外膜蛋白CpnT挑战了这种范例，外膜蛋白CpnT被Mtb用来分泌毒性C末端结构域。该毒素是Mtb在巨噬细胞中复制所需的，是Mtb在巨噬细胞中的主要细胞毒性因子，并引起宿主细胞的坏死性细胞死亡。因此，我们将这种蛋白质命名为结核坏死毒素（TNT）。通过坏死杀死受感染的巨噬细胞是Mtb逃避吞噬体遏制和适应性免疫应答的众所周知但知之甚少的毒力机制。最近，我们发现TNT是一种新的NAD+-糖水解酶，它以ESX-1依赖的方式进入感染的巨噬细胞的胞质溶胶。结核分枝杆菌通过产生抗毒素保护自身免受CpnT/TNT毒性，我们将其命名为TNT免疫因子（IFT）。IFT在结合时使TNT的酶活性失活。IFT的发现使我们能够以mg的量生产TNT蛋白，随后，解决了TNT-IFT复合物的晶体结构，并对纯化的TNT蛋白进行了酶促表征。我们已经确定了无催化活性的TNT突变体，我们将使用这些突变体来确定这种Mtb毒素在最终导致受感染的巨噬细胞坏死的分子途径中的作用。因此，我们现在能够检查一个新的分子机制的Mtb与宿主细胞的相互作用至关重要。这些发现对于200多种具有TNT同源物的细菌和真菌病原体以及理解宿主细胞中NAD+耗尽的分子后果也很重要。