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

项目摘要 细菌病原体对全球健康的威胁越来越大，因为日益严重的 病原菌耐药性。尽管病原体感染宿主的机制多种多样，但 病原菌必须克服的共同障碍是将毒力因子跨多个 膜屏障--包括自身和宿主细胞的。这个过程代表了一种潜在的细菌“阿喀琉斯” 足跟“，用于抑制病机。完成这一壮举的一种强有力的细菌武器是类型IV 分泌系统(T4SS)，根据细菌的不同，由12-30个成分组成的大型复合体， 跨越细菌的内膜和外膜。在革兰氏阴性菌中，这些复合体可以传递 效应器蛋白进入真核细胞，DNA进入其他细菌，和/或毒素进入细菌邻居。我们 纯化和鉴定嗜肺军团菌Dot/ICM的第一个高分辨结构(缺陷在 细胞器运输/细胞内增殖)T4SS的单粒子冷冻电子显微镜(Cryo-EM) 使我们能够建立T4SS组件的原子模型。嗜肺性乳杆菌是一种机会性病原体 感染肺巨噬细胞导致一种名为军团病的潜在致命肺炎 DOT/ICM T4SS是致病所必需的。我们在Dot/ICM T4SS上的工作发现包括 之前未识别的核心T4SS组件的识别、识别和表征 外膜帽(OMC)和周质环(PR)之间的对称性不匹配，以及意外的 OMC中组件的摩尔组织。尽管取得了这些进展，但仍然存在许多问题。该决议 我们的Dot/ICM T4SS结构的高度不够高，无法构建我们密度中所有区域的完整模型 图，我们的纯化明显缺乏在原位冷冻电子断层扫描中看到的主要结构成分 对完整嗜肺乳杆菌的研究，目前还没有关于T4SS是如何从任何 生物体识别、接触和跨膜移动蛋白质。这项建议的目的是 在分子水平上了解Dot/ICM T4SS如何转运蛋白质。为了达到这个目标，我们需要一个 更详细的Dot/ICM T4SS图，保留额外结构的净化复合体的新方法 特征，以及从结构和生化上开始询问底物转运的能力 T4SS。虽然达到这些基准将需要成功完成高风险和具有挑战性的 实验，在这些目标上取得的任何进展都将提供关于分子的有影响力的信息 这种复杂的T4SS的组织，了解T4SS如何调整以转移所需的结果 特定的底物。

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.