Developing a set of robust tools to interrogate Legionella effector function

项目摘要 传染病是全世界人类健康的主要威胁。抗生素的出现 耐药性病原体需要开发新的药物来治疗感染。毒力 病原体用来促进其在宿主细胞中存活和生长的因素代表了 有希望的治疗干预目标。 大多数细菌病原体利用复杂的分泌系统来转移细菌 蛋白质称为效应子进入宿主细胞，调节宿主细胞的过程，为自己的利益。 导致人类致命性肺炎的细菌病原体军团菌， 迄今为止所描述的最大的效应蛋白库之一。分泌缺陷有 对军团菌致病性的多效性作用，因为它们既防止复制的形成， 允许空泡和细菌从降解的溶酶体隔室逃逸。这限制 通过防止它们的增殖同时使宿主细胞能够杀死病原体来减轻细菌负担。 尽管分泌系统本身起着关键作用，但单个效应物的贡献 并且对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主要障碍是缺乏毒力缺陷 与个体效应器的丧失有关。这可能是效应器之间的冗余造成的 或分析有限的宿主细胞类型和/或毒力性状。此外，效应器是 尽管在感染期间许多效应物的协调活动， 一些调节平行或互补的宿主途径，一些成对起作用， 开/关开关和一些调节其他效应器的活动。定义关键事件 负责疾病和控制这些过程的效应物需要高通量 策略，以同时分析各种条件下的整个效应器集合。 所提出的工作的目标是生成一个库L。嗜肺突变体代表 所有384个效应器，在这个过程中，一套遗传工具，系统地询问效应器 规则和功能。总的来说，这些试剂将为许多关键的 调查的途径和研究界的宝贵资源。这样的 研究效应器的系统级方法不仅是前所未有的，而且是至关重要的， 描述了军团菌致病过程中一组关键的毒力因子， 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.