Legionella pneumophila developmental cycle & virulence

性状（由申请方提供）：嗜肺军团菌（L.嗜肺军团菌（pneumophila）作为原生动物的细胞内寄生虫居住在水生生境中，并且当通过气溶胶传播给易感人类时，产生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30年的研究，为什么这种疾病没有在人与人之间传播的解释还没有提供。我们已经在基于细胞的感染模型中记录了一个发育周期，其中营养细菌分化成具有弹性和高度感染性的包囊样代谢休眠形式。这些囊肿对抗生素，洗涤剂和其他有毒物质的影响具有抵抗力，并且可以在水源中长时间存在，从而对健康造成危害。为了确定这些包囊是否是军团病传播的原因，我们提出了以下目标：1）建立原生动物和自来水中的感染模型，以确定促进包囊形态发生的环境条件，其子目标是（i）通过光学和电子显微镜跟踪形态发生;（i）通过报告基因融合;（iii）脉冲追踪放射性标记和蛋白质组分析，以及（iv）评估囊肿形式对清洁剂、杀生物剂和抗生素的适应性;以及对阿米巴原虫和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;和3）通过（i）独特蛋白质的2D凝胶蛋白质组谱分析，（ii）包囊分级分离研究以鉴定外膜、周质和细胞质膜的蛋白质，和（iii）使用基于突变的方法来研究发育调节的基因和调节因子，来表征由原生动物、水和HeLa细胞产生的包囊样形式。我们预期，对军团菌囊肿的持续研究将为我们理解军团菌病的生物学、发病机制、持久性和传播提供重要的新知识。与人类健康的相关性。军团病是全球范围内迅速增长的健康风险，特别是对老年人和免疫功能低下者。从这些研究中获得的知识应有助于改进检测和控制措施，减少对人类的风险。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oxiella，Burkholderia和分枝杆菌属。

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.