HSPGs in Ocular Surface Diseases

描述(申请人提供)：微生物角膜炎是一种威胁视力的感染性疾病，与严重的眼部疾病有关。据估计，每年全球微生物角膜炎的发病率约为50万名患者，仅美国就有3万名患者。由于阻碍微生物定植的解剖屏障，不断清除病原体的机械系统，以及快速建立有效地根除入侵病原体的协调防御反应，眼睛表面通常适合微生物病原体的居住。当这些机制中的一个或几个受到损害，宿主与病原体相互作用的平衡转向有利于致病机制时，就会发生感染。因此，更好地了解宿主-病原体的相互作用是一个主要的公共卫生优先事项，但导致微生物角膜炎发病机制的细菌和宿主机制尚不完全清楚。这项应用的中心目标是定义Syndecan-1，一种主要的细胞表面硫酸肝素蛋白多糖(HSPG)，如何促进细菌性角膜炎的发病。Syndecan-1在角膜上皮细胞中大量表达，但其在角膜中的功能仍不清楚。Syndecan-1既可以作为细胞表面的受体，也可以作为细胞外环境中的可溶性HSPGs，因为它的胞外结构域在一定的病理条件下可以脱落。初步研究表明，Syndecan-1基因缺陷小鼠对金黄色葡萄球菌的角膜感染有明显的抵抗力，提示Syndecan-1是促进金黄色葡萄球菌角膜炎的重要宿主因子。Syndecan-1以这种方式发挥作用的潜在机制尚不清楚，但初步数据也表明，金黄色葡萄球菌诱导角膜上皮细胞脱落Syndecan-1，并以一种依赖于肝素(HS)的方式利用Syndecan-1胞外区的能力来对抗中性粒细胞介导的防御机制。基于这些数据，这项建议将在三个特定的目标上检验细菌对HSPGs的颠覆是细菌性角膜炎发病机制中的一个重要毒力机制的总体假设。目标1将定义Syndecan-1是如何在金黄色葡萄球菌角膜炎中诱导脱落的。目的2阐明Syndecan-1胞外结构域HS促进金黄色葡萄球菌角膜炎的结构特征，目的3将确定Syndecan-1胞外结构域抑制中性粒细胞杀伤金黄色葡萄球菌的生物学机制。拟议的研究将结合细菌性角膜炎的活体模型，使用新的和已建立的遗传、生化和细胞生物学方法。拟议的研究有望揭示先前未知的角膜上皮HSPG的生物学功能，并增加我们对细菌性角膜炎发病机制的分子和细胞过程的了解。

DESCRIPTION (provided by applicant): Microbial keratitis is a sight-threatening infectious disease associated with significant ocular morbidity. By one estimate, the annual incidence of microbial keratitis is approximately 500,000 patients worldwide and 30,000 patients in the US alone. The ocular surface is generally inhabitable for microbial pathogens because of anatomical barriers that impede microbial colonization, mechanical systems that constantly clear pathogens, and coordinated defense responses that are rapidly mounted to effectively eradicate invading pathogens. Infection occurs when one or several of these mechanisms are compromised and the balance of host-pathogen interactions shifts to favor pathogenic mechanisms. Thus, a better understanding of host-pathogen interactions is a major public health priority, but the bacterial and host mechanisms that contribute to the pathogenesis of microbial keratitis are incompletely understood. The central goal of this application is to define how syndecan- 1, a major cell surface heparin sulfate proteoglycan (HSPG), promotes the pathogenesis of bacterial keratitis. Syndecan-1 is abundantly expressed in the corneal epithelium, but much remains unknown of its functions in the cornea. Syndecan-1 functions as receptors on the cell surface and also as soluble HSPGs in the extracellular environment because its extracellular ectodomain can be shed under certain pathological conditions. Preliminary studies showed that mice deficient in syndecan-1 significantly resist corneal infection by Staphylococcus aureus, suggesting that syndecan-1 is an important host factor that promotes S. aureus keratitis. The underlying mechanisms of how syndecan-1 functions in this manner are not understood, but preliminary data also suggested that S. aureus induces syndecan-1 shedding in corneal epithelial cells and exploits the capacity of syndecan-1 ectodomains to counteract neutrophil-mediated defense mechanisms in a heparin sulfate (HS)-dependent manner. Based on these data, this proposal will examine the overall hypothesis that bacterial subversion of HSPGs is an important virulence mechanism in the pathogenesis of bacterial keratitis in 3 Specific Aims. Aim 1 will define how syndecan-1 shedding is induced in S. aureus keratitis. Aim 2 will elucidate the structural features of syndecan-1 ectodomain HS that promote S. aureus keratitis, and Aim 3 will determine the biological mechanism of how syndecan-1 ectodomains inhibit S. aureus killing by neutrophils. The proposed studies will use both novel and established genetic, biochemical, and cell biological approaches in conjunction with in vivo models of bacterial keratitis. The proposed studies are anticipated to uncover previously unknown biological functions of corneal epithelial HSPGs and to increase our understanding of molecular and cellular processes that are central to the pathogenesis of bacterial keratitis.