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.

项目总结/摘要 结核分枝杆菌（Mycobacterium tuberculosis，Mtb）是结核病的病原体，是最致命的病原体之一 几十年来，结核病一直是传染病导致死亡的主要原因。细胞 结核分枝杆菌的包膜在细胞周围形成了一个众所周知的坚韧屏障，保护细菌免受苛刻的试剂 例如抗生素和宿主免疫反应。与此同时，结核病进口营养素 从宿主细胞，如胆固醇，穿过细胞膜。脂质、代谢物和营养物质的运输 穿过细胞膜，在内外膜之间，对于建立和维持细胞膜是至关重要的。 细胞包膜本身，以及细菌生长所需的关键因子的输入。因此，运输系统 促进这种贩运对于允许结核分枝杆菌在其细胞内生态位中生存和繁荣至关重要， 通常是肺部的巨噬细胞 MCE（哺乳动物细胞进入）蛋白家族是转运系统，被认为是 Mtb中的毒力因子，并且与其他毒力因子相比，是分枝杆菌中扩展的蛋白质家族。 双膜细菌在结核分枝杆菌中，几条证据表明MCE系统对于 输入胆固醇和脂肪酸等营养素。E. coli MCE系统已经表明， 它们是锚定在双膜细菌内膜上的多蛋白复合物， 在维持外膜完整性方面起着重要作用，这也提高了这可能 是MCE蛋白在结核分枝杆菌中发挥的作用。本文研究了高复杂MCE系统的结构和机理， 结核分枝杆菌仍然是未知的，研究这些对于了解MCE系统如何工作， 底物是什么，以及它们如何与毒力联系在一起。 该建议的重点是从MCE运输系统的生化和结构表征 结核分枝杆菌和非致病性模型，耻垢分枝杆菌。使用单粒子冷冻EM，质量 光谱，生化和功能测定，我们的目的是研究内源性的结构和功能 Mtb MCE系统，破译哪些蛋白质亚基聚集在一起形成复合物，并定义 蛋白质-蛋白质相互作用对系统的组装和功能很重要。的结果 这项工作将提供重要的见解的结构和功能的大型，多蛋白质MCE复合物， Mtb细胞包膜，以及它们如何影响宿主中的复制和毒力。