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），结核病的病因（TB）是最致命的病原体之一 在地球上，数十年来，结核病一直是由于传染病而导致的死亡原因。细胞 MTB的信封在细胞周围形成一个臭名昭著的障碍物，保护细菌免受刺激性的侵害 在抗生素和宿主免疫反应等环境中。同时，MTB进口营养 从宿主细胞（例如胆固醇）穿过细胞包膜。脂质，代谢物和营养的运输 在整个细胞包膜上，内膜和外膜之间 细胞包络本身，以及进口细菌生长所需的关键因素。因此，运输系统 这有助于这种贩运对于允许MTB在其细胞内生态位中生存和壮成长至关重要，大多数是 通常是肺中的巨噬细胞。 MCE（哺乳动物细胞进入）蛋白质家族是被牵涉到的运输系统 MTB中的毒力因子，与其他 双膜细菌。在MTB中，几条证据表明，MCE系统对 进口营养素，例如胆固醇和脂肪酸。有关大肠杆菌系统系统的最新工作表明 这些是锚定在双膜细菌的内膜的多蛋白络合物，可能 在维持外膜完整性中发挥重要作用 成为MCE蛋白在MTB中起起的作用。高度复杂的MCE系统的结构和机制 MTB仍然未知，研究这些对于了解MCE系统的工作方式至关重要 底物是，以及它们如何与毒力相关。 该建议的重点是MCE运输系统的生化和结构表征 MTB和非致病模型，分枝杆菌Smegmatis。使用单个粒子冷冻EM，质量 光谱法，生化和功能测定法，我们旨在研究内源性的结构和功能 MTB MCE系统，哪些蛋白质亚基组合在一起形成复合物并定义 蛋白质 - 蛋白质相互作用对于系统组装和功能很重要。结果的结果 工作将为大型多蛋白MCE复合物的结构和功能提供重要的见解 MTB细胞包膜，以及它们如何影响宿主中的复制和毒力。