P. aeruginosa type III secreted effectors in corneal disease

描述（由申请人提供）：在美国，铜绿假单胞菌感染是与长时间佩戴隐形眼镜相关的角膜疾病的常见原因。铜绿假单胞菌用于促进疾病的主要毒力因素之一是其III型分泌系统，这是一种分子注射器，允许细菌直接将效应蛋白注射到目标宿主细胞中。III型分泌在角膜疾病中的作用尚未得到广泛研究，但初步报告表明，角膜疾病取决于感染细菌表达的效应体的补体。表达强效磷脂酶ExoU的“细胞毒性”菌株广泛依赖III型分泌来促进疾病，而产生exos的“侵袭性”菌株似乎不依赖III型分泌来引发角膜疾病。我们重新审视了III型分泌在由ExoS+铜绿假单胞菌“侵袭性”分离株引起的角膜疾病发病机制中的作用。我们的初步数据表明，角膜疾病主要依赖于III型分泌，特别是两种效应因子ExoS和ExoT。此外，我们提供的证据表明，III型分泌在角膜疾病中的主要作用是避免通过浸润中性粒细胞杀死。因此，本提案的重点是确定ExoS和ExoT的个体酶活性在体内阻止中性粒细胞清除中的作用。我们将把这些发现与旨在检测exo和ExoT在防止分离的原代中性粒细胞吞噬杀死中的作用的实验联系起来。我们还将研究表达exo的“细胞毒性”菌株和表达exo的“侵袭性”菌株之间的发病机制差异，以确定是否有任何差异与这两种效应物的活性直接相关。了解效应蛋白在角膜疾病中的作用是制定预防和治疗铜绿假单胞菌眼部感染新策略的重要一步。

DESCRIPTION (provided by applicant): P. aeruginosa infections are a common cause of corneal disease associated with extended-wear contact lens use here in the US. One of the primary virulence factors P. aeruginosa uses to promote disease is its type III secretion system, a molecular syringe that allows the bacterium to directly inject effector proteins into targeted host cells. The role of type III secretion in corneal disease has not been studied extensively, but initial reports suggest that corneal disease depends on the complement of effectors being expressed by the infecting bacteria. "Cytotoxic" strains expressing the potent phospholipase ExoU rely extensively on type III secretion to promote disease, whereas ExoS-producing "invasive" strains appear to not rely on type III secretion in order to elicit corneal disease. We have revisited the role of type III secretion in the pathogenesis of corneal disease elicited by ExoS+ "invasive" isolates of P. aeruginosa. Our preliminary data demonstrates that corneal disease relies critically on type III secretion and on the two effectors ExoS and ExoT, in particular. In addition, we provide evidence that the primary role of type III secretion in corneal disease is to stave off killing by infiltrating neutrophils. Accordingly, the focus of this proposal is to determine the role of the individual enzymatic activities of ExoS and ExoT in preventing clearance by neutrophils in vivo. We will correlate these findings with experiments aimed at examining the role of ExoS and ExoT in preventing phagocytic killing by isolated primary neutrophils. We will also examine the difference in pathogenesis between ExoU-expressing "cytotoxic" and ExoS-expressing "invasive" strains of P. aeruginosa in order to determine if any differences can be directly linked to the activities of these two effectors. Understanding the role of effector proteins in corneal disease is an important step towards formulating new strategies for preventing and treating P. aeruginosa infections of the eye. PUBLIC HEALTH RELEVANCE: Pseudomonas aeruginosa is a common cause of eye infections, particularly in contact lens wearers. If left untreated these infections can result in rapid loss of vision. The bacterium injects toxic proteins into host immune cells that normally engulf and kill the invading bacteria. Our research is aimed at understanding how these toxic proteins stave off killing by the host immune system, with the hope that a better understanding of their function will lead to the design of targeted therapies to combat or prevent these infections.