Developing a set of robust tools to interrogate Legionella effector function

项目总结 传染病是世界范围内对人类健康的主要威胁。抗生素的出现 耐药病原体需要开发治疗感染的新药。毒力 病原体用来促进其在宿主细胞中生存和生长的因素代表 有望成为治疗干预的靶点。 大多数细菌病原体使用复杂的分泌系统来转移细菌 被称为效应器的蛋白质进入宿主细胞，为了自身的利益调节宿主细胞的过程。 引起危及人类生命的肺炎的细菌病原体军团菌 迄今为止所描述的最大的效应器蛋白之一。分泌物缺陷有 多效性对军团菌致病作用的影响，因为它们既能防止复制的形成 允许液泡和细菌从降解的溶酶体隔间逸出。这限制了 通过防止细菌的增殖，同时使宿主细胞能够杀死病原体，从而增加细菌的负担。 尽管分泌系统本身起着至关重要的作用，但单个效应器的贡献 军团菌致病的关键事件仍然知之甚少。 确定军团菌如何致病的一个主要障碍是缺乏毒性缺陷 与单个效应器的丧失有关。这可能是由于效应器之间的冗余造成的 或对一组有限的宿主细胞类型和/或毒力特征的分析。此外，效应器是 典型的孤立研究，尽管许多效应器在感染期间协调活动， 通过一些调节的平行或互补的宿主通路，一些成对地发挥作用 开/关开关和一些调节其他效应器活动的开关。定义关键事件 负责疾病和控制这些过程的效应器需要高吞吐量 在各种条件下同时分析整个效应器集合的策略。 这项拟议工作的目标是建立一个嗜肺乳杆菌突变体文库， 所有384个效应器，以及在这个过程中，一套系统询问效应器的遗传工具 规范和功能。总而言之，这些试剂将为无数关键字提供基础 这是调查的途径，是研究界的宝贵资源。这样的一个 系统层面的方法研究效应器不仅是史无前例的，而且是至关重要的 表征军团菌致病过程中的一组关键毒力因素，从而 制定预防和治疗疾病的新战略。

PROJECT SUMMARY Infectious disease is a major threat to human health worldwide. The emergence of antibiotic resistance pathogens necessitates the development of new drugs to treat infection. Virulence factors that pathogens employ to promote their survival and growth in host cells represent promising targets for therapeutic intervention. Most bacterial pathogens employ sophisticated secretion systems to translocate bacterial proteins called effectors into the host cell to modulate host cell processes for their own benefit. The bacterial pathogen Legionella, which causes life-threatening pneumonia in humans, has one of the largest repertoires of effector proteins described to date. Defects in secretion have pleiotropic effects on Legionella pathogenesis as they prevent both the formation of a replication permissive vacuole and bacterial escape from a degradative lysosomal compartment. This limits bacterial burden by preventing their proliferation while enabling pathogen killing by the host cell. Despite the crucial role of the secretion system itself, the contributions of individual effectors and the critical events responsible for Legionella pathogenesis remain poorly understood. A major obstacle in defining how Legionella causes disease is the lack virulence defects associated with loss of individual effectors. This can result from redundancy between effectors or the analysis of a limited set of host cell types and/or virulence traits. Moreover, effectors are typically studied in isolation despite the coordinated activities of many effectors during infection, with some modulating parallel or complementary host pathways, some functioning in pairs as on/off switches and some regulating the activity of other effectors. Defining critical events responsible for disease and the effectors governing these processes requires a high throughput strategy to simultaneously analyze the entire collection of effectors under a variety of conditions. The goal of the proposed work is to generate a library L. pneumophila mutants representing all 384 effectors and, in the process, a set of genetic tools to systematically interrogate effector regulation and function. Collectively, these reagents will provide a foundation for numerous key avenues of investigation and an invaluable resource to the research community. Such a systems level approach to studying effectors is not only unprecedented but paramount to characterizing a critical set of virulence factors in Legionella pathogenesis and thus the development of new strategies to prevent and treat disease.