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。P. aeruginosa利用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，从而引起严重感染。目前鉴定的四种效应蛋白分别是ExoU、ExoS、ExoT和ExoY。在人类和动物急性肺炎模型中，ExoU和exo与严重疾病的关系最为密切。有趣的是，大多数临床分离株分泌ExoU或exo，但不是两者都分泌，这表明它们在发病机制中起着功能相似或冗余的作用。了解这两种酶上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。

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.