Legionella pneumophila developmental cycle & virulence

描述(由申请人提供)：嗜肺军团菌(L.hamphila)作为原生动物的一种细胞内寄生虫存在于水生栖息地，当通过气雾剂传播给易受影响的人类时，会产生一种称为军团病的急性肺炎。这种疾病的传播总是单向的--从环境到人类，尽管有30年的研究，但还没有对这种疾病为什么不会在人与人之间传播提供解释。我们已经记录了基于细胞的感染模型的发育周期，在该模型中，营养细菌分化为囊状代谢休眠形式，具有弹性和高度传染性。这些包囊对抗生素、洗涤剂和其他有毒物质的影响具有抵抗力，并可长期存在于水源中，对健康构成危害。为了确定这些包囊是否与军团病的传播有关，我们提出了以下目标：1)在原生动物和自来水中建立感染模型，以确定促进包囊形态发生的环境条件，子目标是(I)通过光学和电子显微镜跟踪包囊的形态发生；(I)通过报告基因融合；(Iii)脉冲追逐放射性标记和蛋白质组图谱；(Iv)评估包囊形态对洗涤剂、杀生剂和抗生素的抵抗力；以及对阿米巴和A/J小鼠巨噬细胞的感染性。2)在A/J小鼠气溶胶攻击模型中评估包囊的毒力；以及3)通过(I)二维凝胶蛋白质组图谱寻找独特蛋白质，(Ii)包囊分离研究以确定外膜、周质和细胞质膜的蛋白质，以及(Iii)使用基于突变的方法来研究发育调控基因和调控因子，来表征来自原生动物、水和HeLa细胞的包囊形态。我们预计，对浮游孢囊的持续研究将有助于我们对退伍军人病的生物学、发病机制、持久性和传播的了解。与人类健康相关。退伍军人病是一种全球范围内迅速增长的健康风险，尤其是对老年人和免疫功能低下的人。从这些研究中获得的知识应该有助于改进检测和控制措施，降低对人类的风险。这些研究还应提供与其他机会性环境病原体相关的见解，包括奥希氏菌、伯克霍尔德氏菌和分枝杆菌。

DESCRIPTION (provided by applicant): Legionella pneumophila (L. pneumophila) resides in aquatic habitats as an intracellular parasite of protozoa and when transmitted in aerosols to susceptible humans, produces an acute pneumonia known as Legionnaires' disease. Transmission of this disease is always one-way - from the environment to humans, and despite 30 years of research, an explanation for why this disease is not spread from person to person has not been offered. We have documented a developmental cycle in cell based infection models in which vegetative bacteria differentiate into cyst-like metabolically dormant forms that are resilient and highly infectious. These cysts are resistant to the effects of antibiotics, detergents and other toxic agents and can persist in water sources for extended periods where they present a health hazard. To determine if these cysts are responsible for transmission of Legionnaires' disease we propose the following aims: 1) To develop infection models in protozoa and in tap water to identify environmental conditions that promote cyst morphogenesis with sub aims of (i) tracking morphogenesis by light and electron microscopy; (i) by reporter gene fusions; (iii) pulse chase radiolabeling and proteome profiling and (iv) to assess cyst forms for resilience to detergents, biocides, and antibiotics; and infectiousness for amoebae and A/J mouse macrophages. 2) Assessment of virulence of cysts in an A/J mouse aerosol challenge model; and 3) To characterize the cyst like forms arising from protozoa, water and HeLa cells by (i) 2D gel proteome profiling for unique proteins, (ii) cyst fractionation studies to identify proteins of the outer membrane, periplasm and cytoplasmic membrane and (iii) use a mutation based approach to study developmentally regulated genes and regulatory factors. We anticipate that continued study of the planktonic cysts will contribute significant new knowledge to our understanding of the biology, pathogenesis, persistence and transmission of Legionnaires' disease. Relevance to human health. Legionnaires' disease is a rapidly growing health risk world wide, and especially for the elderly and immunocompromised. The knowledge gained from these studies should lead to improved detection and control measures that reduce the risk to humans. These studies should also provide insights relevant to other opportunistic environmental pathogens including oxiella, Burkholderia and species of Mycobacterium.