An enabling technology to dissect critical molecular events in bacterial pathogenesis

一种剖析细菌发病机制中关键分子事件的技术

• 批准号: 9316213
• 负责人: Tamara O'Connor
• 金额: $ 24.49万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-23 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9316213
• 关键词: Address; Aerosols; Affinity; Alveolar Macrophages; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Proteins; Binding; Biochemical; Biological Assay; Biological Models; Breathing; Cell physiology; Cells; Chemicals; Chimeric Proteins; Communicable Diseases; Cytolysis; DNA Sequence Alteration; Detection; Development; Disease; Event; Foundations; Genetic; Goals; Growth; Health; Host Defense; Human; Impairment; Individual; Infection; Legionella; Legionella pneumophila; Life; Location; Mammalian Cell; Mass Spectrum Analysis; Mediating; Methodology; Methods; Molecular; Monitor; Organelles; Outcome; Parasites; Pathogenesis; Pathogenicity; Pathway interactions; Phenotype; Play; Population; Process; Proteins; Regulation; Research; Role; Signal Transduction; Specificity; System; Techniques; Technology; Testing; Therapeutic Intervention; Vacuole; Variant; Virulence; Virulence Factors; Water; antimicrobial drug; base; cell type; contaminated water; crosslink; falls; innovation; insight; interest; killings; loss of function; macrophage; man; microorganism; new technology; new therapeutic target; novel strategies; novel therapeutic intervention; novel therapeutics; overexpression; pathogen; pathogenic bacteria; prevent; protein protein interaction

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. A fundamental challenge in developing antibiotics is that many pathogens replicate inside host cells rendering them inaccessible to antimicrobial agents. Critical processes for pathogen growth in host cells represent the most promising new targets for therapeutic intervention. Thus, a systematic strategy to identify virulence mechanisms central to establishing growth within the host cell and avoiding killing by host defenses is paramount for defining new targets for therapeutic intervention. Through specialized secretion systems, many intracellular pathogens deploy an arsenal of proteins termed effectors that modulate numerous host cell processes to establish growth. Loss of function of the secretion machinery restricts pathogen survival and replication demonstrating a critical role for effectors in disease. Despite the importance of effectors, very little is known about the events that determine when individual effectors are deployed or their individual contributions during infection. This can largely be attributed to the inability to monitor effector populations in host cells due to low endogenous levels that are undetectable by standard biochemical techniques and redundancy amongst effectors whereby loss of any individual effector does not result in a discernible phenotype. Innovative approaches that overcome the limitations of classic genetic and biochemical approaches are necessary to further our understanding of effector-mediated virulence mechanisms employed by pathogens. BioID is a powerful biochemical technique used to define protein-protein interactions. The goal of the proposed research is to adapt BioID to examine the interactions of bacterial virulence proteins in the context of an infection. Using Legionella pathogenesis as a model system, we will apply this technology to define molecular events central to effector translocation, targeting and function. Our BioID-based experimental system will allow several key questions central to the infection process to be addressed that cannot be resolved using currently available methodologies: 1) What are the host targets of individual effectors? 2) How are effectors distributed once they enter the host cell? 3) When are individual effectors secreted and into which host cell types? 4) What are the signals that trigger effectors selection for secretion? Once established, this technology will be broadly applicable to the study of a variety of pathogenic microorganisms and provide a foundation for defining critical events in disease that is crucial to developing new strategies for therapeutic intervention.

项目摘要 传染病是全世界人类健康的主要威胁。的出现 抗生素抗性病原体需要开发新的药物来治疗感染。一 开发抗生素的根本挑战是许多病原体在宿主体内复制， 使它们无法接触到抗微生物剂。病原体的关键过程 在宿主细胞中的生长代表了治疗干预的最有希望的新靶点。因此，在本发明中， 一个系统的战略，以确定毒力机制的核心建立增长内 宿主细胞和避免被宿主防御杀死对于定义新的靶点是至关重要的， 治疗干预 通过专门的分泌系统，许多细胞内病原体部署了一个兵工厂， 调节许多宿主细胞过程以建立生长的称为效应子的蛋白质。损失 分泌机制的功能限制了病原体的存活和复制， 效应子在疾病中的关键作用。尽管效应器的重要性， 关于确定何时部署单个效应器或其单个效应器的事件 感染期间的贡献。这在很大程度上可以归因于无法监测效应器 由于标准品检测不到的内源性水平较低， 生物化学技术和效应子之间的冗余， 效应子不会导致可辨别的表型。创新办法，克服 经典的遗传和生化方法的局限性是必要的，以进一步我们的研究。 了解病原体采用的效应子介导的毒力机制。 BioID是一种强大的生物化学技术，用于定义蛋白质-蛋白质相互作用。的 拟议研究的目标是调整BioID来检查细菌之间的相互作用， 毒力蛋白在感染的情况下。以军团菌致病为模型 系统，我们将应用这项技术来定义分子事件的核心效应易位， 目标和功能。我们基于BioID的实验系统将允许几个关键问题 要解决的感染过程的核心问题，目前无法使用 可用的方法：1）什么是个别效应器的主机目标？2)好吗 效应子进入宿主细胞后会如何分布3)个体效应物何时分泌， 植入哪种宿主细胞4)哪些信号触发效应子选择分泌？ 一旦建立，这项技术将广泛适用于各种研究。 病原微生物，并为定义疾病中的关键事件提供基础， 对于开发新的治疗干预策略至关重要。