Corneal infection: bacterial localization versus virulence

 描述(申请人提供)：配戴隐形眼镜会使健康的角膜容易受到感染，最常见的是革兰氏阴性细菌铜绿假单胞菌。对角膜感染发病机制的研究大多采用绕过上皮屏障的划痕损伤模型，这阻碍了我们对细菌与其相互作用如何影响发病机制的理解。我们的实验室最近开发了一种表面损伤模型和成像方法，首次能够在整个小鼠眼球内对单个定植细菌进行亚细胞定位。此前，角膜上皮细胞/细菌相互作用的细节只能在培养细胞中进行研究，培养细胞不一定模拟体内感染。铜绿假单胞菌编码一个三型分泌系统(T3SS)，这是一种将毒素注入宿主细胞细胞质的分子注射器。使用培养的细胞，这个实验室已经证明了大多数铜绿假单胞菌角膜分离株分泌T3SS毒素EXOS，并将它们命名为“侵袭性菌株”，因为它们可以在培养的上皮细胞内复制。在小鼠角膜感染中，它们也可以通过透射电子显微镜在角膜上皮细胞内看到，即使在使用膜非渗透性抗生素治疗后也可以恢复。培养的细胞研究表明，EXOS可以将质膜重塑为细菌在其中复制的气泡利基。虽然含有细胞内细菌的水泡也会在体内感染期间出现(使用新的实时成像策略)，但侵袭发生的频率、时间和地点，细胞内细菌是否在天然角膜上皮内复制，以及这些现象是否/如何参与发病机制仍有待确定。假说是，对于侵袭性菌株，在原位定植角膜上皮的细菌在所有三个细胞层都成为细胞内，细胞内定位激活细菌T3SS，T3SS激活使细胞内复制。支持这一点的未发表数据显示，T3SS有助于小鼠角膜内的上皮遍历，而我使用培养细胞收集的数据显示，T3SS只有在细菌内化后才被触发，后者是一种范式转变。这次培训机会将使我能够通过提供活体细菌、动物模型、最先进的成像技术和体内毒力因子调节方面的经验，在细菌毒素生物化学方面奠定基础。该项目将探索铜绿假单胞菌内化的意义，同时确定T3SS如何促进角膜上皮的穿越。鉴于上皮穿越是感染的早期步骤，这项研究可能导致在病理过程开始之前预防感染的策略。

 DESCRIPTION (provided by applicant): Contact lens wear can predispose the healthy cornea to infection, most commonly by the gram-negative bacterium Pseudomonas aeruginosa. Research into the pathogenesis of corneal infection has mostly used a scratch injury model that bypasses the epithelial barrier, hindering our understanding of how bacterial interactions with it influence pathogenesis. Our lab recently developed a superficial injury model and imaging methods that for the first time enables subcellular localization of individual colonizing bacteria within whole mouse eyeballs. Previously, details of corneal epithelial cell/bacterial interactions could only be studied in cultured cells, which do not necessarily mimic in vivo infections. P. aeruginosa encodes a Type Three Secretion System (T3SS), a molecular syringe that injects toxins into host cell cytoplasm. Using cultured cells, this lab has shown that most P. aeruginosa corneal isolates of secrete the T3SS toxin ExoS, and named them "invasive strains," because they can replicate inside cultured epithelial cells. In mouse corneal infections, they can also be seen inside cells of the corneal epithelium using transmission electron microscopy, and can be recovered even after treatment with membrane non-permeable antibiotics. Cultured cell studies show that ExoS can remodel the plasma membrane into a bleb niche wherein bacteria replicate. While blebs containing intracellular bacteria also occur during infection in vivo (seen using the new live imaging strategy), how frequently, when, and where invasion occurs, whether intracellular bacteria replicate within native corneal epithelium, and if/how these phenomena contribute to pathogenesis is to be established. The hypothesis is that for an invasive strain, a significant percentage of bacteria colonizing the corneal epithelium in situ become intracellular in all three cellular layers, that intracellular localization activates the bacterial T3SS, and tha activation of the T3SS enables intracellular replication. Unpublished data supporting this show that the T3SS contributes to epithelial traversal within the mouse cornea, while data I collected using cultured cells show the T3SS is triggered only after bacteria are internalized, the later being a paradigm shift. Using the in situ mouse eye model, Aim 1 will determine bacterial distribution during corneal epithelial traversal, including intra/extracellular spaces and subcellular localization, Aim 2 will examine the role of T3SS activation and delivery of exotoxins into epithelial cell cytoplasm, while Aim 3 will determine sites of intracellular replication and ue mutant P. aeruginosa lacking T3SS components to determine their contribution. This training opportunity will allow me to build on a foundation in bacterial toxin biochemistry by providing experience with live bacteria, animal models, state of the art imaging technologies, and virulence factor regulation in vivo. The project will explore the significance of P. aeruginosa internalization, while determining how the T3SS contributes to traversal of the corneal epithelium. Given that epithelial traversal is an early step in infection, this research could leadto strategies for preventing infection before pathological processes are initiated.