Type II Secretion and Legionella Pneumophila Infection

描述(由申请人提供)：嗜肺军团菌(LP)是军团病的主要病原体，军团病是一种常见的、可能致命的肺炎形式。这种细菌在天然和人造水系统中无处不在，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，感染水生原生动物、巨噬细胞或上皮细胞。以前，我们确定LP具有II型分泌系统(T2S)，并假设T2S分泌的蛋白是环境持久性、细胞内感染和毒力的媒介。在上一次资助期间，我们记录了T2S是阿米巴、肺泡巨噬细胞和肺上皮细胞内感染以及细菌在实验感染小鼠肺内持续存在所必需的。然后结合LP上清液的蛋白质组学分析和LP基因组的电子分析，揭示T2S输出的广泛性质。除了我们确认的预测活性外，还发现了与已知蛋白没有相似性的“新的”外源蛋白。利用缺乏一个或多个依赖于T2S的外源蛋白(效应物)的突变体，我们确定金属蛋白酶(ProA)和核糖核酸酶(SRNA)促进阿米巴感染，磷脂酶C(PlcA/B)和几丁质酶(Chia)促进哺乳动物细胞感染，而一种新的蛋白(Lpg0264)促进阿米巴和巨噬细胞感染。体内竞争分析随后确定了四个似乎促进肺内LP存活的T2S效应物；即Chia、PlcA/B、Lpg0264和一种类似于ASTIN的蛋白酶(LegP)。此外，我们还发现LP在琼脂表面表现出一种依赖于T2S的聚集表型。总之，LP T2S对细胞内感染、成群、毒力以及水中的低温生存都是唯一关键的，它比任何其他T2S系统阐述更多的效应因子和编码更广泛的活动。在目前的方案中，我们将使用遗传学和各种感染模型和细胞生物学工具来i)确定一些新的“新”效应器对细胞内感染的重要性，ii)确定依赖T2S的成群对细胞内感染的重要性，iii)确定ProA、sRNA、PlcA/B和Lpg0264影响的细胞内位置和运输模式，以及iv)确认PlcA/B、Lpg0264和LegP这三种新的新型效应器的重要性，以及成群在肺炎小鼠模型中的作用。拟议的研究将：1)显著增加我们对LP发病机制的了解，LP是美国和世界各地的一个重要的公共卫生问题；2)扩大我们对细菌蛋白质分泌和细胞内感染的分子理解；3)对利用或预测使用T2S的其他重要的环境病原体产生影响；以及4)为疾病诊断、治疗或预防提供潜在的新靶点。公共卫生相关性：我们发现，嗜肺军团菌的所谓II型分泌系统促进了哺乳动物宿主细胞(如巨噬细胞和上皮细胞)和实验感染小鼠肺部的细菌生长。因此，我们假设细菌通过该系统分泌的蛋白质(效应物)是毒力因子，因此是疾病诊断或预防的潜在靶点。为了验证这一假设，我们将i)使用细胞内感染的体外模型和肺炎的动物模型来表征缺乏特定效应物的细菌突变体，以确定那些促进感染的效应物，以及ii)监测那些被证明对感染重要的蛋白质在细胞内的表达模式。

DESCRIPTION (provided by applicant): Legionella pneumophila (Lp) is the primary agent of Legionnaires' disease, a common and potentially fatal form of pneumonia. The bacterium 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, macrophages, or epithelia. Previously, we determined that Lp possesses a type II secretion (T2S) system and hypothesized that proteins secreted by T2S are mediators of environmental persistence, intracellular infection, and virulence. During the last grant period, we documented that T2S is required for intracellular infection of amoebae, alveolar macrophages, and lung epithelia as well as bacterial persistence in the lungs of experimentally-infected mice. Proteomic analysis of Lp supernatants and in silico analysis of the Lp genome were then combined to reveal the extensive nature of the T2S output. In addition to predicted activities, which we confirmed, "novel" exoproteins were identified that had no similarity to known proteins. Using mutants lacking one or more T2S-dependent exoproteins (effectors), we determined that a metalloprotease (ProA) and ribonuclease (SrnA) promote amoeba infection, phospholipases C (PlcA/B) and a chitinase (ChiA) facilitates infection of mammalian cells, and a novel protein (Lpg0264) promotes both amoebal and macrophage infection. In vivo competition assays then identified four T2S effectors that appear to promote Lp survival in lungs; i.e., ChiA, PlcA/B, Lpg0264, and an astacin-like protease (LegP). Additionally, we discovered that Lp exhibits a swarming phenotype on agar surfaces that is dependent upon T2S. In sum, Lp T2S is uniquely critical for intracellular infection, swarming, virulence, as well as low-temperature survival in water, elaborating more effectors and encoding a wider variety of activities than is appreciated for any other T2S system. In the current proposal, we will use genetics and various infection models and cell biological tools to i) determine the importance of a select number of new "novel" effectors for intracellular infection, ii) determine the importance of T2S-dependent swarming for intracellular infection, iii) determine the intracellular location of and trafficking patterns influenced by ProA, SrnA, PlcA/B, and Lpg0264, and iv) confirm the importance of PlcA/B, Lpg0264, and LegP, the new novel effectors, and swarming in a murine model of pneumonia. The proposed studies will i) increase significantly our understanding of the pathogenesis of Lp, which is an important 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, environmental pathogens that utilize or are predicted to use T2S, and iv) offer potential new targets for disease diagnosis, treatment, or prevention. PUBLIC HEALTH RELEVANCE: We have discovered that the so-called type II secretion system of Legionella pneumophila promotes bacterial growth in mammalian host cells (e.g., macrophages and epithelial cells) and in the lungs of experimentally infected mice. We therefore hypothesize that the proteins (effectors) secreted by the bacterium through this system are virulence factors and therefore are potential targets for disease diagnosis or prevention. To test this hypothesis, we will i) characterize bacterial mutants that are lacking specific effectors using in vitro models of intracellular infection and an animal model of pneumonia in order to identify those effectors that promote infection, and ii) monitor the intracellular expression patterns of those proteins that are shown to be important for infection.