P. aeruginosa type III secreted effectors in corneal disease

描述(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。铜绿假单胞菌用来促进疾病的主要毒力因素之一是它的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入目标宿主细胞。III型分泌物在角膜疾病中的作用尚未得到广泛研究，但最初的报道表明，角膜疾病依赖于感染细菌表达的效应物的补充。表达有效磷脂酶ExoU的“细胞毒性”菌株广泛依赖III型分泌物来促进疾病，而产生EXOS的“侵袭性”菌株似乎不依赖III型分泌物来引发角膜疾病。我们回顾了III型分泌物在EXOS+“侵袭性”铜绿假单胞菌引起的角膜疾病发病机制中的作用。我们的初步数据表明，角膜疾病严重依赖于III型分泌物，特别是两种效应物EXOS和ExoT。此外，我们提供的证据表明，III型分泌物在角膜疾病中的主要作用是通过渗透中性粒细胞来延缓杀戮。因此，这项建议的重点是确定EXOS和ExoT的单个酶活性在防止中性粒细胞在体内清除中的作用。我们将把这些发现与旨在检测EXOS和ExoT在防止分离的初级中性粒细胞吞噬杀伤中的作用的实验相关联。我们还将检查表达ExoU的“细胞毒性”和表达EXOS的“侵袭性”铜绿假单胞菌之间的致病机制差异，以确定是否有任何差异可以直接与这两个效应器的活性有关。了解效应蛋白在角膜疾病中的作用是制定预防和治疗眼部铜绿假单胞菌感染的新策略的重要一步。 公共卫生相关性：铜绿假单胞菌是眼部感染的常见原因，尤其是在隐形眼镜佩戴者中。如果不及时治疗，这些感染可能会导致快速失明。这种细菌将有毒蛋白质注入宿主免疫细胞，宿主免疫细胞通常会吞噬并杀死入侵的细菌。我们的研究旨在了解这些有毒蛋白是如何抵御宿主免疫系统的杀戮的，希望更好地了解它们的功能将导致设计靶向疗法来对抗或预防这些感染。

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.