An in vivo Gene Deletion System for Analyzing Temporal Requirement of the Dot/Icm

用于分析 Dot/Icm 时间需求的体内基因删除系统

• 批准号: 7472176
• 负责人: Zhao-Qing Luo
• 金额: $ 7.28万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-08 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7472176
• 关键词: Accounting; Address; Animal Model; Bacteria; Bacterial Counts; Bacterial Genes; Bacterial Proteins; Carrier Proteins; Cells; Complex; Development; Disruption; Essential Genes; Gene Deletion; Gene Family; Gene Targeting; Genes; Genetic; Goals; Host Defense Mechanism; Infection; Infectious Agent; Isopropyl Thiogalactoside; Legionella pneumophila; Legionnaires' Disease; Light; Lytic; Methods; Microbial Biofilms; Numbers; Personal Satisfaction; Pneumonia; Population; Protein Secretion; Protein translocation; Proteins; Public Health; Research; Role; Staging; System; Systems Analysis; Techniques; Thinking; Time; Vacuole; base; design; in vivo; mutant; novel; numb protein; pathogen; permissiveness; uptake

DESCRIPTION (provided by applicant): Legionella pneumophila, the causative agent of Legionnaires' disease transfers a large number of bacterial proteins into host cells via a specialized protein translocation system termed Dot/Icm to construct an intracellular compartment that supports bacterial multiplication. Earlier studies suggest that the Dot/Icm protein transporter is required only for minutes during infection for the establishment the replicative vacuole. However, due to technique limitation other possibilities that can account for this observation have never been addressed. Moreover, recent progress in studies of the function of Dot/Icm strongly suggests that this transporter is required considerably longer than previously thought. For example, some effectors are transferred into the infected cells throughout the infection cycle and effectors required for non-lytic release of the bacteria from the host at the terminal end of the infection have been identified. In this proposal, we plan to reexamine the temporal requirement of the Dot/Icm system by L. pneumophila by using a novel genetic system that allows us to specifically disrupt target gene(s) as any time after bacterial uptake. We have successfully designed a Cre/loxP-based inducible in vivo gene deletion strategy suitable for examining the temporal requirement of specific protein(s). Following bacterial uptake by host cells, we will use IPTG to induce the deletion of gene(s) essential for the function of the Dot/Icm transporter. Function of the transporter then will be assessed by its ability to translocate protein substrates into host cells and bacterial multiplication will be analyzed by the formation of replicative vacuoles as well as by total bacterial counts. These studies should allow us to more thoroughly analyze the temporal requirement of the Dot/Icm pathogenic machinery by the pathogen during infection. Furthermore, the described genetic setup should provide technique support for study whether a specific bacterial protein or protein complex is important for maintaining a development or infection status. PUBLIC HEALTH RELEVANCE: Infection by Legionella pneumophila often leads to the development of a fatal form of pneumonia. In addition, study on this bacterium could share light on our understanding of other intracellular pathogens because L. pneumophila and these infectious agents share many pathogenic mechanisms.

描述（由申请人提供）：肺炎军团菌是Lecionnaires疾病的致病药物，通过专门的蛋白质易位系统将大量的细菌蛋白转移到宿主细胞中，称为DOT/ICM，以构建一个支撑细菌倍增的细胞内圆形室。较早的研究表明，在感染期间，DOT/ICM蛋白转运蛋白仅需要在几分钟内才能建立复制液泡。但是，由于技术的限制，其他可以解释此观察结果的可能性从未得到解决。此外，对DOT/ICM功能的研究的最新进展强烈表明，该转运蛋白的要求比以前想象的要长得多。例如，在整个感染周期中，某些效应子被转移到感染细胞中，并且已经确定了在感染的末端从宿主中非散散释放细菌所需的效应子。在此提案中，我们计划通过使用一种新型的遗传系统来重新检查点/ICM系统的时间需求，该系统使我们能够在细菌摄取后的任何时候都可以特异性地破坏靶基因。我们已经成功设计了一种基于CRE/LOXP的体内基因缺失策略，适合检查特定蛋白质的时间需求。宿主细胞的细菌摄取后，我们将使用IPTG诱导DOT/ICM转运蛋白功能必不可少的基因缺失。然后，转运蛋白的功能将通过其将蛋白质底物转移到宿主细胞中的能力进行评估，细菌繁殖将通过复制液泡的形成以及总细菌计数来分析。这些研究应使我们能够更彻底地分析感染过程中病原体DOT/ICM致病机制的时间需求。此外，所描述的遗传设置应为​​研究提供技术支持，是否对于维持发育或感染状态是否重要。 公共卫生相关性：肺炎军团菌的感染通常会导致肺炎的致命形式。此外，对该细菌的研究可能会阐明我们对其他细胞内病原体的理解，因为肺炎乳杆菌和这些传染性剂具有许多致病机制。