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Exploring protein translocation by the Legionella pneumophila Dot/Icm Type IV Section System

探索嗜肺军团菌 Dot/Icm IV 型切片系统的蛋白质易位

基本信息

批准号：
10426349
负责人：
Melanie Diane Ohi
金额：
$ 19.5万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-11 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10426349
关键词：
Affinity Affinity Chromatography Alveolar Macrophages Antibiotic Resistance Bacteria Bacterial Proteins Benchmarking Biochemical Biochemistry Biological Assay Cells Complex Coupling Cryo-electron tomography Cryoelectron Microscopy Cytoplasm DNA Defect Detergents Development Drug resistance Environment Eukaryotic Cell Genetic Goals Gram-Negative Bacteria In Situ In Vitro Infection Inhalation Intracellular Transport Legionella pneumophila Legionnaires&apos Disease Lipids Lung Lysosomes Mammalian Cell Maps Mass Spectrum Analysis Membrane Microbial Biofilms Modeling Molecular Organelles Organism Pathogenesis Phagosomes Pneumonia Process Protein Analysis Protein translocation Proteins Protocols documentation Resolution Specificity Speed Structural Models Structure System Testing Toxin Type III Secretion System Pathway Type IV Secretion System Pathway Vesicle Virulence Factors War Work arm density experimental study global health high risk host colonization improved macrophage molecular modeling opportunistic pathogen particle pathogen pathogenic bacteria periplasm physical process preservation resistance gene success three dimensional structure weapons

项目摘要

Project Summary Bacterial pathogens represent an increasing threat to global health due to the growing problem of pathogen drug resistance. Although the mechanisms deployed by pathogens to infect hosts are diverse, a common obstacle that pathogenic bacteria must overcome is moving virulence factors across multiple membrane barriers – both their own and the host cell’s. This process represents a potential bacterial “Achilles heel” for inhibiting pathogenesis. One potent bacterial weapon that accomplishes this feat is the Type IV Secretion System (T4SS), a large complex, composed of 12-30 components depending on the bacteria, that spans the bacterial inner and outer membranes. In Gram-negative bacteria these complexes can deliver effector proteins into eukaryotic cells, DNA into other bacteria, and/or toxins into bacterial neighbors. We purified and determined the first high-resolution structure of the Legionella pneumophila Dot/Icm (defect in organelle transport/ intracellular multiplication) T4SS using single particle cryo-electron microscopy (cryo-EM) allowing us to build atomic models of T4SS components. L. pneumophila is an opportunistic pathogen that infects lung macrophages leading to a potentially fatal pneumonia called Legionnaires’ Disease and the Dot/Icm T4SS is required for pathogenesis. Discoveries from our work on the Dot/Icm T4SS include the identification of a previously unrecognized core T4SS component, identification and characterization of symmetry mismatches between the outer membrane cap (OMC) and periplasmic ring (PR), and an unexpected molar organization of components in the OMC. Despite this progress, many questions remain. The resolution of our Dot/Icm T4SS structure was not high enough to build a complete model of all the regions in our density map, our purification clearly lacks major structural components seen in in situ cryo-electron tomography studies of intact L. pneumophila, and there is currently no molecular understanding for how the T4SS from any organism identifies, engages, and moves proteins across membranes. The purpose of this proposal is to understand on a molecular level how the Dot/Icm T4SS translocates proteins. To reach this goal we need a more detailed map of the Dot/Icm T4SS, new ways to purify the complex that preserve additional structural features, and the ability to begin structurally and biochemically interrogating substrate translocation by the T4SS. While reaching these benchmarks will require the successful completion of high-risk and challenging experiments, progress made on any of these goals will provide impactful information about the molecular organization of this complex T4SS, results required for understanding how T4SSs are tuned to translocate specific substrates.

项目总结