The role of Pseudomonas aeruginosa ExoS during early pneumonia.

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。目前鉴定的四种效应蛋白是ExoU、ExoS、ExoT和ExoY。ExoU和ExoS与急性肺炎的人类和动物模型中的严重疾病最密切相关。有趣的是，大多数临床分离株分泌ExoU或ExoS，但不是两者都分泌，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使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治疗方法奠定基础。 本项目旨在了解ExoS在肺炎中的致病机制。初步数据显示，分泌ExoS的菌株以高数量持续存在于肺中，而基因的缺失导致在感染的最初24小时内从肺中快速清除。ExoS[+]菌株能够持续存在，尽管引起嗜中性粒细胞和巨噬细胞向肺的强烈募集，表明ExoS以某种方式使这些细胞失能。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防止铜绿假单胞菌从肺部清除。使用急性肺炎的小鼠模型和优化的基于FRET的报告基因测定，将鉴定在急性肺炎的早期阶段注射ExoS的细胞类型。将通过检查吞噬细胞的细菌内化、这些细胞对细菌的杀伤和吞噬细胞存活来确定早期肺炎期间ExoS中毒对吞噬细胞的影响。各种体外和体内试验，包括基于荧光的内化试验，将用于研究对吞噬细胞的这种影响。这些目标将定义ExoS的细胞靶点以及在早期肺炎期间ExoS中毒对这些细胞的影响。这些信息，结合目前对ExoU机制的了解，将有助于更好地了解感染期间铜绿假单胞菌的总体发病机制。

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.