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的病原体，是医院获得性肺炎的主要原因。为了引起严重的感染，铜绿假单胞菌使用III型分泌系统将效应蛋白直接注入真核细胞的细胞质中。当前鉴定的四种效应蛋白是Exou，Exos，Exot和Exoy。在急性肺炎的人和动物模型中，Exou和Exos与严重疾病最密切相关。有趣的是，大多数临床分离株分泌了外观，但并非两者都表明它们在发病机理上起功能相似或冗余作用。了解这两种酶上不同的毒素如何允许铜绿假单胞菌引起严重的肺炎，将为预防和治疗铜绿假单胞菌感染而奠定基础。 该项目旨在了解肺炎EXOS的致病机制。初步数据表明，肺部分泌菌株的持续数量较高，而基因的缺失导致最初24小时感染的肺部迅速清除。尽管将中性粒细胞和巨噬细胞募集到肺部，但Exos [+]菌株仍然能够持续存在，这表明Exos以某种方式使这些细胞无能为力。该项目将确定EXOS是否通过在肺炎早期损害吞噬细胞来阻止肺中的铜绿假单胞菌清除。使用急性肺炎的小鼠模型和优化的基于FRET的记者测定法，将鉴定出在急性肺炎早期注射外观的细胞类型。外源中毒对肺炎早期吞噬细胞的影响将通过检查吞噬细胞的细菌内在化，这些细胞杀死细菌以及吞噬细胞的存活来定义。各种体外和体内测定（包括基于荧光的内在化测定法）将用于研究对吞噬细胞的影响。这些目的将定义EXOS的细胞靶标以及在肺炎早期在这些细胞上与Exos中毒的后果。此类信息以及当前关于Exou机制的知识将使人们更好地了解感染过程中铜绿假单胞菌的总体发病机理。