The role of Pseudomonas aeruginosa ExoS during early pneumonia.

铜绿假单胞菌 ExoS 在早期肺炎中的作用。

• 批准号: 8259505
• 负责人: Stephanie M Rangel
• 金额: $ 2.81万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8259505
• 关键词: A Mouse; ADP Ribose Transferases; Actins; Acute; Acute Pneumonia; Animal Model; Bacteria; Biological Assay; Cell Death; Cells; Clinical; Cytoplasm; Cytoskeleton; Data; Disease; ERM protein; Eukaryotic Cell; Fluorescence; Fluorescence Resonance Energy Transfer; Foundations; Frequencies; Future; GTPase-Activating Proteins; Gene Deletion; Genes; Goals; Human; In Vitro; Infection; Injection of therapeutic agent; Integration Host Factors; Intoxication; Knowledge; Lead; Link; Lung; Measures; Molecular; Morbidity - disease rate; Necrosis; Neutrophil Infiltration; Nosocomial pneumonia; Pathogenesis; Phagocytes; Phagocytosis Inhibition; Phospholipase; Play; Pneumonia; Proteins; Pseudomonas aeruginosa; Recruitment Activity; Reporter; Role; Severities; Severity of illness; Staging; Tertiary Protein Structure; Testing; Therapeutic; Toxin; Type III Secretion System Pathway; United States; base; cell killing; cell type; cellular targeting; design; in vitro Assay; in vivo; interest; killings; macrophage; mortality; mouse model; pathogen; prevent; research study

Pseudomonas aeruginosa is a gram-negative pathogen that is the major cause of hospital-acquired pneumonia. To cause serious infections, P. aeruginosa uses a type III secretion system to directly inject effector proteins into the cytoplasm of eukaryotic cells. The four effector proteins currently identified are ExoU, ExoS, ExoT, and ExoY. ExoU and ExoS have been most closely linked to severe disease in human and animal models of acute pneumonia. Interestingly, most clinical isolates secrete either ExoU or ExoS but not both, suggesting that they play functionally similar or redundant roles in pathogenesis. Understanding how these two enzymatically different toxins both allow P. aeruginosa to cause severe pneumonia will lay the foundation to develop therapeutics to prevent and treat P. aeruginosa infections. This project aims to understand the pathogenic mechanism of ExoS in pneumonia. Preliminary data show that ExoS-secreting strains persist in the lungs at high numbers whereas deletion of the gene results in rapid clearance from the lung over the initial 24 hr of infection. ExoS[+] strains are able to persist despite eliciting robust recruitment of neutrophils and macrophages to the lung, suggesting that ExoS somehow incapacitates these cells. This project will determine if ExoS acts to prevent P. aeruginosa clearance from the lungs by impairing phagocytes during early pneumonia. Using a mouse model of acute pneumonia and an optimized FRET-based reporter assay, the cell types injected with ExoS during the early stages of acute pneumonia will be identified. The effect of ExoS intoxication on phagocytes during early pneumonia will be defined by examining bacterial internalization by phagocytes, killing of bacteria by these cells, and phagocyte survival. Various in vitro and in vivo assays, including a fluorescence-based internalization assay, will be used to study this effect on phagocytes. These aims will define the cellular targets of ExoS and the consequences of intoxication with ExoS on these cells during early pneumonia. Such information, in conjunction with the current knowledge on the mechanisms of ExoU, will lead to a better understanding of the overall pathogenesis of P. aeruginosa during infection.

铜绿假单胞菌是一种革兰氏阴性病原体，是医院获得性肺炎的主要原因。为了引起严重感染，铜绿假单胞菌使用III型分泌系统将效应蛋白直接注射到真核细胞的细胞质中。目前确定的四种效应蛋白是 ExoU、ExoS、ExoT 和 ExoY。 ExoU 和 ExoS 与人类和动物急性肺炎模型中的严重疾病关系最为密切。有趣的是，大多数临床分离株分泌 ExoU 或 ExoS，但不会同时分泌两者，这表明它们在发病机制中发挥功能相似或冗余的作用。了解这两种酶促不同的毒素如何导致铜绿假单胞菌引起严重肺炎，将为开发预防和治疗铜绿假单胞菌感染的疗法奠定基础。 该项目旨在了解ExoS在肺炎中的致病机制。初步数据显示，分泌 ExoS 的菌株在肺部大量存在，而删除该基因会导致在感染的最初 24 小时内迅速从肺部清除。尽管引起中性粒细胞和巨噬细胞向肺部大量募集，ExoS[+]菌株仍能够持续存在，这表明ExoS以某种方式使这些细胞丧失能力。该项目将确定 ExoS 是否通过在早期肺炎期间损害吞噬细胞来阻止铜绿假单胞菌从肺部清除。使用急性肺炎小鼠模型和基于 FRET 的优化报告分析，将鉴定在急性肺炎早期阶段注射 ExoS 的细胞类型。早期肺炎期间 ExoS 中毒对吞噬细胞的影响将通过检查吞噬细胞的细菌内化、这些细胞对细菌的杀伤以及吞噬细胞的存活来确定。各种体外和体内测定，包括基于荧光的内化测定，将用于研究这种对吞噬细胞的影响。这些目标将确定 ExoS 的细胞靶点以及早期肺炎期间 ExoS 中毒对这些细胞的影响。这些信息与目前对 ExoU 机制的了解相结合，将有助于更好地了解铜绿假单胞菌感染期间的整体发病机制。