Developing a set of robust tools to interrogate Legionella effector function

开发一套强大的工具来询问军团菌效应器功能

• 批准号: 10306407
• 负责人: Tamara O'Connor
• 金额: $ 8.19万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-11-23 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10306407
• 关键词: Address; Alveolar; Antibiotic Resistance; Bacterial Proteins; Binding; Biochemical; Cell physiology; Cells; Cloning; Collection; Communicable Diseases; Communities; Complex; DNA cassette; Defect; Deletion Mutation; Detection; Development; Disease; Environment; Event; Foundations; Gene Deletion; Gene Expression; Generations; Genetic; Genetic Transcription; Goals; Growth; Health; Human; Immune system; Individual; Infection; Insertional Mutagenesis; Investigation; Legionella; Legionella pneumophila; Libraries; Life; Lung; Massive Parallel Sequencing; Measures; Methods; Molecular; Monitor; Morphology; Mutation; Parasites; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Phenotype; Philadelphia; Physiological; Physiology; Pigments; Plasmids; Pneumonia; Procedures; Process; Production; Proteins; Protocols documentation; Reagent; Regulation; Reporter; Research; Resources; Role; Site; System; Techniques; Therapeutic Intervention; Vacuole; Virulence; Virulence Factors; Work; base; cell motility; cell type; genetic manipulation; genomic locus; high throughput screening; mutant; novel therapeutics; null mutation; pathogen; pathogenic bacteria; pleiotropism; prevent; response; screening; targeted treatment; tool; trait; transposon sequencing

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.

项目概要 传染病是全世界人类健康的主要威胁。抗生素的出现 耐药病原体需要开发新药来治疗感染。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 治疗干预的有希望的目标。 大多数细菌病原体利用复杂的分泌系统来转移细菌 蛋白质被称为效应子进入宿主细胞，为了自身利益而调节宿主细胞的过程。 细菌病原体军团菌会导致人类致命的肺炎， 迄今为止描述的最大的效应蛋白库之一。分泌缺陷有 对军团菌发病机制的多效性影响，因为它们阻止复制的形成 允许液泡和细菌从降解的溶酶体室中逃逸。这限制了 通过防止细菌增殖同时使宿主细胞杀死病原体来减轻细菌负担。 尽管分泌系统本身起着至关重要的作用，但个体效应器的贡献 而导致军团菌发病机制的关键事件仍然知之甚少。 定义军团菌如何引起疾病的一个主要障碍是缺乏毒力缺陷 与个体效应器的损失有关。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 尽管许多效应器在感染过程中协调活动，但通常是孤立研究的， 具有一些平行或互补的调节宿主途径，一些成对发挥作用 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器需要高通量 在各种条件下同时分析整个效应器集合的策略。 拟议工作的目标是生成代表嗜肺军团菌突变体的文库 所有 384 个效应器，以及在此过程中的一套遗传工具来系统地询问效应器 调节和功能。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。这样一个 研究效应器的系统级方法不仅是前所未有的，而且对于 表征军团菌发病机制中一组关键的毒力因子，从而 制定预防和治疗疾病的新策略。