Type II Secretion and Legionella pneumophila Infection

描述(由申请人提供)：嗜肺军团菌(LP)是军团病的病原体，军团病是一种常见的、可能致命的肺炎。肺炎在天然和人造水系统中普遍存在，吸入受污染的气雾剂后会感染肺部。在水和肺中，LP以细胞内寄生虫的形式生长，感染水生原虫或肺泡巨噬细胞。在此之前，我们证明了Lp表达一种II型分泌(T2S)系统，该系统介导&GT；25蛋白(底物)的分泌，并且是阿米巴和哺乳动物宿主细胞以及小鼠肺最佳感染所必需的。在上一次资助期间，我们记录了分泌型plac(一种酰基转移酶)、ProA(一种金属蛋白酶)、sRNA(一种核糖核酸酶)、NTTA和Lpw18401，以及另外两种“新”底物以一种独特的宿主特异性方式促进棘阿米巴、哈特曼尼拉和奈格勒阿米巴的细胞内生长和存活。我们还证明了T2S和一组新的底物促进了肺巨噬细胞内的细胞内生长/存活，并且可能是通过帮助将宿主蛋白Rab1保留在含有军团菌的空泡上来实现的。最后，在其他结果中，我们记录了T2S及其至少两种底物，即ProA和NTTA，抑制了感染的巨噬细胞的趋化因子和细胞因子的输出，这一过程很可能会阻碍LP从感染的肺中清除。这种对细胞因子产生的颠覆部分是由于抑制了信号转导和细胞因子基因转录。总而言之，我们一直处于证明T2S在LP生命周期及其相关疾病的所有主要方面都是关键的前沿，比目前任何其他细菌T2S系统所了解的更多底物和编码更广泛的活动。在目前的提案中，我们将确定i)PLAC、ProA、SRNA和NTTA和其他新型T2S底物促进LP在多个阿米巴中生长的机制，这些底物是LP疾病从水系统传播到人类的关键环节；ii)T2S及其新型底物Lpw02811和Lpw18401如何调节巨噬细胞中的Rab1，巨噬细胞是LP感染宿主的关键宿主细胞；以及iii)T2S及其底物包括NTTA调节LP感染巨噬细胞中的细胞因子信号的机制，以及评估细胞因子颠覆对LP感染肺中细胞炎症反应的影响。拟议的研究将：1)显著增加我们对LP发病机制的了解，LP是美国和世界各地日益重要的公共卫生问题，II)扩大我们对细菌蛋白质分泌和细胞内感染的分子理解，III)对利用或预测使用T2S的其他重要病原体有影响，IV)对由于是巨噬细胞内寄生虫而致病的其他细菌有影响，以及v)为疾病诊断、治疗或预防提供潜在的新靶点。

DESCRIPTION (provided by applicant): Legionella pneumophila (Lp) is the agent of Legionnaires' disease, a common and potentially fatal form of pneumonia. Lp is ubiquitous in natural and man-made water systems and infects the lungs after the inhalation of contaminated aerosols. In water and the lungs, Lp grows as an intracellular parasite, infecting either aquatic protozoa or alveolar macrophages. Previously, we demonstrated that Lp expresses a type II secretion (T2S) system that mediates the secretion of >25 proteins (substrates) and is required for optimal infection of both amoebal and mammalian host cells and the murine lung. During the last grant period, we documented that secreted PlaC (an acyltransferase), ProA (a metalloprotease), SrnA (a ribonuclease), NttA, and Lpw18401, and two other "novel" substrates promote intracellular growth/survival in Acanthamoeba, Hartmannella, and Naegleria amoebae in a unique host-specific fashion. We also demonstrated that T2S and a subset of novel substrates enhance intracellular growth/survival within lung macrophages and likely do so by helping to retain host protein Rab1 on the Legionella-containing vacuole. Finally, among other results, we documented that T2S and at least two of its substrates, i.e., ProA and NttA, dampen the chemokine and cytokine output of infected macrophages, a process that would most likely impede the clearance of Lp from the infected lung. This subversion of cytokine production was due, in part, to dampened signal transduction and cytokine gene transcription. In sum, we have been at the forefront of demonstrating that T2S is critical in all of the major facets of the Lp lifecycle and its associated disease, elaborating more substrates and encoding a wider variety of activities than is currently appreciated for any other bacterial T2S system. In the current proposal, we will now determine i) mechanisms by which PlaC, ProA, SrnA, and NttA and other novel T2S substrates enhance Lp growth in multiple amoebae, which are the essential links in Lp disease transmission from water systems to humans, ii) how T2S and its novel substrates Lpw02811 and Lpw18401 act to modulate Rab1 in macrophages, which are the critical host cell in the Lp-infected mammalian host, and iii) the mechanisms by which T2S and its substrates, including NttA, modulate cytokine signaling in Lp-infected macrophages as well as assess the impact of cytokine subversion on the cellular inflammatory response in the Lp-infected lung. The proposed studies will i) increase significantly our understanding of the pathogenesis of Lp, which is an important and increasing public health concern within the US and throughout the world, ii) expand our molecular understanding of both bacterial protein secretion and intracellular infection, iii) have implications for other important pathogens that utilize or are predicted to use T2S, iv) have implications for other bacteria that are pathogenic by virtue of being intracellular parasites of macrophages, and v) offer potential new targets for disease diagnosis, treatment, or prevention.