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

项目摘要 细菌性病原体对全球健康构成越来越大的威胁，这是由于日益严重的细菌感染问题。 病原体耐药性尽管病原体感染宿主的机制多种多样， 病原菌必须克服的一个常见障碍是将毒力因子在多个 膜屏障--包括它们自己的和宿主细胞的。这一过程代表了潜在的细菌“阿基里斯” 用于抑制发病。完成这一壮举的一种强大的细菌武器是IV型 分泌系统（T4 SS），一个大的复合体，由12-30个组分组成，取决于细菌， 跨越细菌的内外膜。在革兰氏阴性细菌中，这些复合物可以递送 效应蛋白进入真核细胞，DNA进入其他细菌，和/或毒素进入细菌邻居。我们 纯化并确定了嗜肺军团菌Dot/Icm的第一个高分辨率结构（ 细胞器运输/细胞内增殖）T4 SS，使用单粒子冷冻电子显微镜（冷冻EM） 使我们能够建立T4 SS组件的原子模型。L.嗜肺菌是一种机会致病菌， 感染肺巨噬细胞，导致一种潜在的致命性肺炎，称为军团病， Dot/Icm T4 SS是发病所必需的。我们在Dot/Icm T4 SS上的工作发现包括： 识别以前未识别的核心T4 SS组件，识别和表征 外膜帽（OMC）和周质环（PR）之间的对称性不匹配，以及意想不到的 OMC中组分的摩尔组织。尽管取得了这些进展，但仍存在许多问题。该决议 我们的Dot/Icm T4 SS结构的密度不足以建立我们密度中所有区域的完整模型 图中，我们的纯化明显缺乏在原位低温电子断层扫描中看到的主要结构成分 完整L.嗜肺菌，目前还没有分子理解如何从任何T4 SS 生物体识别、接合和移动蛋白质穿过膜。这项建议的目的是 在分子水平上理解Dot/Icm T4 SS如何易位蛋白质。为了实现这一目标，我们需要 Dot/Icm T4 SS的更详细的地图，纯化复合物的新方法，保留额外的结构 特征，以及开始结构和生化询问底物易位的能力， T4SS虽然达到这些基准需要成功完成高风险和具有挑战性的工作 在这些目标上取得的任何进展都将提供有关分子生物学的有影响力的信息。 组织这个复杂的T4 SS，结果需要了解T4 SS是如何调整易位 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.