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，军团病是一种常见且可能致命的肺炎形式。Lp在自然和人造水系统中无处不在，吸入被污染的气溶胶后会感染肺部。在水和肺中，Lp作为细胞内寄生虫生长，感染水生原生动物或肺泡巨噬细胞。先前，我们证明了Lp表达II型分泌（T2S）系统，该系统介导bbb25蛋白（底物）的分泌，并且是阿米巴原虫和哺乳动物宿主细胞以及小鼠肺的最佳感染所必需的。在上一个资助期间，我们记录了分泌的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以及其他两种“新型”底物以独特的宿主特异性方式促进棘阿米巴原虫、Hartmannella阿米巴原虫和Naegleria阿米巴原虫的细胞内生长/存活。我们还证明，T2S和一组新型底物可以增强肺巨噬细胞内的细胞内生长/存活，这可能是通过帮助在含有军团菌的液泡上保留宿主蛋白Rab1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，抑制被感染巨噬细胞的趋化因子和细胞因子的输出，这一过程很可能阻碍被感染肺中Lp的清除。细胞因子生产的这种颠覆部分是由于抑制了信号转导和细胞因子基因转录。总之，我们已经站在了证明T2S在Lp生命周期及其相关疾病的所有主要方面都至关重要的前沿，与目前任何其他细菌T2S系统相比，我们阐述了更多的底物并编码了更广泛的活动。在当前的建议,现在我们将决定我温馨的)机制,快速三角帆船,SrnA, NttA和其他小说t2基质增强Lp增长多个变形虫,必要的链接在Lp从水系统对人类疾病传播,2)如何t2及其小说基质Lpw02811 Lpw18401行为调节Rab1在巨噬细胞,这是至关重要的宿主细胞Lp-infected哺乳动物宿主,和iii) t2的机制及其基板,包括NttA,在lp感染的巨噬细胞中调节细胞因子信号，并评估细胞因子颠覆对lp感染肺细胞炎症反应的影响。拟议的研究将i)显著增加我们对Lp发病机制的理解，这是美国和全世界一个重要且日益增加的公共卫生问题，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)对其他利用或预计使用T2S的重要病原体有意义。Iv)由于是巨噬细胞的细胞内寄生虫，对其他致病性细菌有启示，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.