Type II Secretion and Legionella pneumophila Infection

描述(申请人提供)：嗜肺军团菌(Lp)是引起退伍军人病肺炎的病原体。在其水生栖息地，LP以原生动物的细胞内寄生虫的形式生存，接种到肺部后，它在肺泡巨噬细胞内繁衍生息。此前，我们发现了一个与PilD同源的Lp基因(PilD)，在其他革兰氏阴性菌中，它介导了菌毛的生物发生和II型蛋白的分泌。事实上，一个LP PilD突变体缺少菌毛。更重要的是，PilD突变体，而不是Pilin突变体，对于阿米巴和巨噬细胞的感染是有缺陷的，这表明LP具有促进感染的II型分泌系统。在最后一次授予期间，我们证实LP有一个II型系统(LSP)，它介导包括新型酶在内的许多蛋白质的分泌，并且对原生动物和人类细胞的感染都是至关重要的。我们的其他一些数据表明，LP肽基-Pro异构酶MIP和SurA在分泌过程中起作用。目前，LP是唯一已知的研究细胞内病原体II型分泌的系统。在上一次授予期间，我们还证明了LSP突变体在A/J小鼠肺部的生存能力受到了极大的损害，其缺陷的严重性表明LPII型分泌物是一个关键的毒力决定因素，可能不仅仅与巨噬细胞感染有关。最后，我们进行了新的观察，发现LSP是在12-25℃生长所必需的。因此，LPII型分泌物对于细胞内感染、毒力和低温生长是唯一关键的。在目前的提案中，我们的目标是识别对细胞内感染至关重要的II型外源蛋白，确认MIP和SurA在分泌中的作用，并确定II型分泌如何促进体内存活以及在水和阿米巴中的低温生长。这些研究的结果将增加我们对LP生理和发病机制的了解，ii)为细菌蛋白质分泌、细胞内感染和低温生长提供新的见解，以及III)对其他重要的人类病原体，包括其他细胞内寄生虫和细胞外病原体，如弧菌、假单胞菌和伯克霍尔德氏菌，具有低温生存和II型系统的影响。

DESCRIPTION (provided by applicant): Legionella pneumophila (Lp) is the agent of Legionnaires' disease pneumonia. In its aquatic habitat, Lp survives as an intracellular parasite of protozoans, and after inoculation into the lung, it flourishes within alveolar macrophages. Previously, we discovered an Lp gene (pilD) whose product is homologous with PilD, which, in other Gram-negatives, mediates pilus biogenesis and type II protein secretion. Indeed, an Lp pilD mutant lacked pili. More importantly, the pilD mutant, but not a pilin mutant, was defective for infection of amoebae and macrophages, suggesting that Lp has a type II secretion system that promotes infection. During this last grant period, we confirmed Lp has a type II system (lsp) that mediates secretion of many proteins, including novel enzymes, and is critical for infection of both protozoa and human cells. Some of our other data suggested that the Lp peptidyl-prolyl isomerase Mip and SurA have a role in the secretion process. Presently, Lp is the only known system for studying type II secretion in an intracellular pathogen. In the last grant period, we also demonstrated that lsp mutants are greatly impaired for survival in the lungs of A/J mice, with the severity of their defect indicating that Lp type II secretion is a key virulence determinant that may be involved in more than just macrophage infection. Finally, we made the novel observation that lsp is required for growth at 12-25 degrees C. Thus, Lp type II secretion is uniquely critical for intracellular infection, virulence, and low-temperature growth. In the current proposal, we aim to identify the type II exoproteins that are critical for intracellular infection, confirm the role of Mip and SurA in secretion, and determine how type II secretion promotes in vivo survival as well as low-temperature growth in water and amoebae. The results of these studies will i) increase our understanding of Lp physiology and pathogenesis, ii) provide new insight into bacterial protein secretion, intracellular infection, and low-temperature growth, and iii) have implications for other important human pathogens, including both other intracellular parasites and extracellular pathogens, such as Vibrio, Pseudomonas, and Burkholderia that survive at low-temperature and have type II systems.