Structural characterization of MCE transport systems from Mycobacterium tuberculosis

结核分枝杆菌 MCE 转运系统的结构表征

• 批准号: 10681871
• 负责人: Gira Bhabha
• 金额: $ 81.68万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-03 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10681871
• 关键词: Antibiotics; Architecture; Bacteria; Binding; Biochemical; Biological Assay; Cause of Death; Cells; Cellular Membrane; Cholesterol; Communicable Diseases; Complex; Complex Analysis; Cryoelectron Microscopy; Development; Drug resistance; Environment; Escherichia coli; Fatty Acids; Genes; Genus Mycobacterium; Gram-Negative Bacteria; Growth; Hydrophobicity; Immune response; Immune system; In Vitro; Individual; Learning; Link; Lipids; Lung; Macrophage; Maintenance; Mammalian Cell; Mass Spectrum Analysis; Membrane; Modeling; Multiprotein Complexes; Mutation; Mycobacterium smegmatis; Mycobacterium tuberculosis; Nutrient; Operon; Pathogenesis; Phagosomes; Planets; Play; Protein Family; Protein Subunits; Proteins; Proteomics; Role; Structure; System; Testing; Time; Tuberculosis; Virulence; Virulence Factors; Work; cell envelope; combat; design; gene product; insight; lipid transport; membrane biogenesis; mycobacterial; particle; pathogen; polypeptide; protein complex; protein function; protein protein interaction; protein structure; protein transport; resistant strain; structural biology; trafficking

Project Summary/Abstract Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is one of the deadliest pathogens on the planet, and for decades, TB has been the leading cause of death due to infectious disease. The cell envelope of Mtb forms a notoriously tough barrier around the cell, protecting the bacterium from harsh agents in the environment such as antibiotics and host immune responses. At the same time, Mtb imports nutrients from the host cell, such as cholesterol, across the cell envelope. Transport of lipids, metabolites and nutrients across the cell envelope, between the inner and outer membranes is critical for building and maintaining the cell envelope itself, and for import of key factors required for bacterial growth. Therefore, the transport systems that facilitate this trafficking are critical for allowing Mtb to survive and thrive in its intracellular niche, most typically macrophages in the lungs. The MCE (Mammalian Cell Entry) family of proteins are transport systems that have been implicated as virulence factors in Mtb, and are an expanded protein family in mycobacteria compared with other double-membraned bacteria. In Mtb, several lines of evidence suggest that MCE systems are important for importing nutrients such as cholesterol and fatty acids. Recent work on E. coli MCE systems has shown that these are multi-protein complexes anchored in the inner membrane of double-membraned bacteria, and may play an important role in the maintenance of outer membrane integrity, raising the possibility that this may also be a role that MCE proteins play in Mtb. The structure and mechanisms of the highly complex MCE systems in Mtb remain unknown, and studying these is critical for understanding how MCE systems work, what their substrates are, and how they may be linked to virulence. This proposal is focused on biochemical and structural characterization of MCE transport systems from Mtb and the non-pathogenic model, Mycobacterium smegmatis. Using single particle cryo-EM, mass spectrometry, biochemical and functional assays, we aim to study the structure and function of endogenous Mtb MCE systems, decipher which protein subunits come together to form complexes and define protein-protein interactions that are important for the systems to assemble and function. The results of this work will provide important insights into the structure and function of the large, multi-protein MCE complexes in the Mtb cell envelope, and how they influence replication and virulence in the host.

项目摘要/摘要 结核分枝杆菌(Mtb)是引起结核病的病原体，是最致命的病原体之一。 在这个星球上，几十年来，结核病一直是传染病导致的主要死亡原因。单元格 结核分枝杆菌的包膜在细胞周围形成了一道众所周知的坚硬屏障，保护细菌免受刺激性物质的伤害。 在环境中，如抗生素和宿主免疫反应。同时，结核分枝杆菌还进口营养物质。 从宿主细胞，如胆固醇，穿过细胞膜。脂类、代谢物和营养素的运输 穿过细胞被膜，在内膜和外膜之间对于建立和维持 细胞外膜本身，以及细菌生长所需的关键因子的进口。因此，运输系统 为这种贩运提供便利是让结核分枝杆菌在其细胞内利基市场生存和繁荣的关键，大多数 典型的肺内巨噬细胞。 MCE(哺乳动物细胞进入)家族蛋白是一种运输系统，被认为是 Mtb中的毒力因子，与其他分枝杆菌相比，它是分支杆菌中一个扩大的蛋白质家族 双膜细菌。在MTB，有几条证据表明，MCE系统对 进口胆固醇和脂肪酸等营养素。最近关于大肠杆菌MCE系统的研究表明， 这些是锚定在双膜细菌内膜上的多蛋白质复合体，可能 在维持外膜完整性方面发挥重要作用，增加了这也可能 可能是MCE蛋白在结核分枝杆菌中的作用。高度复杂的MCE系统的结构和机制 MTB仍然是未知的，研究这些对于理解MCE系统如何工作、它们的 底物是，以及它们如何与毒力联系在一起。 这项建议的重点是MCE运输系统的生化和结构特征 MTB和非致病模型污垢分枝杆菌。使用单粒子低温电磁，质量 通过光谱分析、生化分析和功能分析，我们旨在研究内源蛋白的结构和功能。 MTB MCE系统，破译哪些蛋白质亚单位聚集在一起形成复合体并定义 蛋白质之间的相互作用对系统的组装和功能非常重要。这样做的结果 这项工作将为了解大的、多蛋白质的MCE复合体的结构和功能提供重要的见解 结核分枝杆菌细胞膜，以及它们如何影响宿主的复制和毒力。