Structure-function analysis of type IVB secretion systems

摘要 嗜肺军团菌是一种革兰氏阴性细菌，在环境中的原生动物体内复制，但 也可以在肺泡巨噬细胞内复制，从而导致一种类似肺炎的疾病，称为 退伍军人症。嗜肺性乳杆菌能够在正常杀菌能力范围内存活和复制 通过改变吞噬细胞的内吞途径，阻止吞噬小体-溶酶体的快速融合。L. 嗜肺性肺炎通过向宿主体内注入大量毒力因子来扰乱正常宿主细胞的功能 细胞质通过IVB型分泌系统(T4BSS)。嗜肺性乳杆菌T4BSS由20- 7个DOT/ICM基因，编码细菌细胞膜中的一个大分子复合体。这个 DOT/ICM系统是细胞内生长和毒力必不可少的系统。相比之下，大多数人 由于多个水平的冗余，~300Dot/ICM底物对这两个性状似乎都是必不可少的。 因此，嗜肺乳杆菌Dot/ICM代表了最可行的治疗干预靶点。 尽管已经获得了大量关于组件和功能的信息 DOT/ICM系统，许多问题仍然存在。这项提案将是最近在以下方面取得的一些进展的后续 Dot/ICM T4BSS。其中包括发现Dot/ICM系统位于 需要通过这种专门的分泌系统进行细菌和底物的极性输出，才能 细胞内靶向LCV(含空泡的军团菌)。第二个主要的进步是 DotL与分泌伴侣结合的c-末端结构的测定。决赛 先进的是通过低温电子显微镜获得了这种分泌器的第一张图像。总体来说，这项研究 在几个方面与人类健康有关。首先，建立嗜肺性乳杆菌用于 在巨噬细胞内存活和复制将为进一步了解它是如何导致疾病的，并可能 揭示用于药物治疗的新靶点。第二，由于专门的分泌系统 常用多种细菌病原体，了解嗜肺乳杆菌分泌物 该装置很可能适用于理解其他细菌致病的分子机制 病原体，可作为预防或治疗多种不同疾病的基础。

Abstract Legionella pneumophila is a Gram-negative bacteria that replicates inside protozoa in the environment but can also replicate within alveoloar macrophages, thereby causing a pneumonia-like disease called Legionnaires’ disease. L. pneumophila is able to survive and replicate within normally bactericidal phagocytic cells by altering their endocytic pathway and preventing rapid phagosome-lysosome fusion. L. pneumophila perturbs normal host cell function by injecting a large number of virulence factors into the host cell cytoplasm via a type IVB secretion system (T4BSS). The L. pneumophila T4BSS is encoded by twenty- seven dot/icm genes, which encode a large macromolecular complex in the bacterial cell envelope. The Dot/Icm system is absolutely required for intracellular growth and virulence. In contrast, the majority of the ~300 Dot/Icm substrates appear to be dispensable for both traits due to multiple levels of redundancy. Therefore, the L. pneumophila Dot/Icm represents the most viable target for therapeutic intervention. Although a significant amount of information has been acquired about the assembly and function of the Dot/Icm system, many questions remain. This proposal will follow-up on a number of recent advances on the Dot/Icm T4BSS. These include the discovery that the Dot/Icm system is located at the poles of the bacterium and polar export of substrates by this specialized secretion system is required for proper intracellular targeting of the LCV (Legionella containing vacuole). The second major advance was the determination of the structure of the c-terminus of DotL bound to the secretion chaperones. The final advance was the acquisition of the first image of this secretion apparatus by cryoEM. Overall this research relates to human health in several ways. First, establishing the mechanism used by L. pneumophila to survive and replicate inside macrophages will provide additional insight into how it causes disease and may reveal novel targets to be used for drug therapy. Second, since specialized secretion systems are commonly used by a variety of bacterial pathogens, knowledge gained about the L. pneumophila secretion apparatus is likely to be applicable to understanding the molecular mechanisms of virulence used by other pathogens, and could serve as the basis to prevent or treat a number of different diseases.