Type II Secretion and Legionella pneumophila Infection

描述（由申请方提供）：嗜肺军团菌（Lp）是军团菌性肺炎的病原体。在其水生栖息地，Lp作为原生动物的细胞内寄生虫存活，并且在接种到肺中后，其在肺泡巨噬细胞内寄生。在此之前，我们发现了一个Lp基因（PilD），其产物与PilD同源，在其他革兰氏阴性菌中，PilD介导菌毛生物发生和II型蛋白分泌。事实上，Lp pilD突变体缺乏皮利。更重要的是，pilD突变体，而不是菌毛蛋白突变体，是有缺陷的感染阿米巴和巨噬细胞，这表明Lp有一个II型分泌系统，促进感染。在这最后一个赠款期间，我们证实了Lp有一个II型系统（lsp），介导许多蛋白质的分泌，包括新的酶，是至关重要的原生动物和人类细胞的感染。我们的一些其他数据表明，Lp肽基脯氨酰异构酶Mip和SurA在分泌过程中发挥作用。目前，Lp是唯一已知的用于研究细胞内病原体中的II型分泌的系统。在最后一个资助期，我们还证明了lsp突变体在A/J小鼠肺中的生存能力大大受损，其缺陷的严重程度表明Lp II型分泌是一个关键的毒力决定因素，可能不仅仅参与巨噬细胞感染。最后，我们进行了新的观察，lsp是在12-25摄氏度生长所必需的。因此，Lp II型分泌对于细胞内感染、毒力和低温生长是唯一关键的。在目前的建议中，我们的目标是确定的II型外蛋白是细胞内感染的关键，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生存以及在水和变形虫的低温生长。这些研究的结果将i）增加我们对Lp生理学和发病机制的理解，ii）提供对细菌蛋白分泌、细胞内感染和低温生长的新见解，和iii）对其他重要的人类病原体具有影响，包括其他细胞内寄生虫和细胞外病原体，如在低温下存活并具有II型系统的弧菌、假单胞菌和伯克霍尔德氏菌。

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.