Cytoskeletal Keratins in Epithelial Immunity to Bacterial Keratitis

描述(由申请人提供)：细胞骨架角蛋白在细菌性角膜炎的上皮免疫中角膜感染是全世界成年人和儿童视力受损和失明的主要原因。由于目前的治疗方法不足以预防视力丧失，因此需要替代的治疗策略。在盖茨基金会大挑战探索基金资助的一项研究中，我们探索了为什么角膜表面在清除接种到其表面上的细菌方面非常有效。这项研究导致我们发现了一类新的抗菌肽(KDAMPs)，它是角蛋白6A的富含甘氨酸的C末端片段，角蛋白6A是一种主要的中间丝蛋白，存在于多种上皮细胞的细胞骨架中。与大多数已知的抗菌肽(AMPs)不同，KDAMP具有耐盐性，其杀灭作用不依赖于多肽/细菌膜的静电吸引、多肽的疏水性和特定的二级结构，这表明它们与其他抗菌肽的作用方式不同。初步数据还显示，角蛋白6A的敲除显著增加了细菌对完整小鼠角膜的黏附。该项目的总体目标是了解KDAMP的产生和抗菌活性的机理和功能方面。假设胞浆角蛋白6A的内源性蛋白分解过程产生角蛋白衍生抗菌肽(KDAMPs)，其单体的磷酸化促进角蛋白细丝周转增加KDAMPs的生成和活性，KDAMPs通过裂解细菌膜杀死细菌，KDAMPs保护角膜免受细菌黏附。这将在三个目标中得到检验。目标1将确定KDAMP是如何通过鉴定涉及的内源性蛋白酶以及磷酸化诱导的角蛋白增溶在调节KDAMP的水平和活性中的作用而产生的 KDAMPS。目的2通过研究KDAMPs与细菌膜的结构相互作用，以及通过鉴定其在主要角膜病原体铜绿假单胞菌上的细菌受体，来探讨KDAMPs杀菌的机制。目的3研究KDAMPs在保护角膜免受铜绿假单胞菌感染中的作用。这将通过在眼表定位KDAMPs，并确定它们在角蛋白6A介导的抑制细菌黏附和随后的角膜感染敏感性中的贡献来完成。结合FPLC的蛋白酶体和蛋白酶抑制剂将被用于鉴定产生KDAMPs的蛋白酶。将使用磷酸酶抑制剂、激酶激活剂和细菌抗原来促进角蛋白的溶解。孔的形成和多肽/膜的相互作用将通过脂质体和固体核磁共振波谱进行研究。细菌受体将通过细胞分级和免疫沉淀进行鉴定。采用免疫印迹、酶联免疫吸附试验和特制抗体免疫染色等方法检测KDAMPs。这项研究的结果将提高我们对健康的角膜如何抵抗感染的理解，并可能导致控制角膜和其他部位感染的新的治疗策略。

DESCRIPTION (provided by applicant): Cytoskeletal Keratins in Epithelial Immunity to Bacterial Keratitis Corneal infection is a major cause of visual impairment and blindness in both adults and children worldwide. Since current treatments are inadequate to prevent vision loss, alternative therapeutic strategies are needed. In a study funded by a Gates Foundation Grand Challenges Explorations grant, we explored why the corneal surface is exquisitely effective at clearing bacteria that are inoculated onto its surface. That study led us to the discovery of a novel class of antimicrobial peptides (KDAMPs), which are glycine-rich C-terminal fragments of keratin 6A, a major intermediate filament protein found in the cytoskeleton of a wide range of epithelial cells. In contrast to most known antimicrobial peptides (AMPs), KDAMPs are salt-tolerant and their killing action is independent of peptide/bacterial membrane electrostatic attraction, peptide hydrophobicity and defined secondary structure, suggesting they differ from other AMPs in how they interact with bacterial membranes. Preliminary data also showed that knockdown of keratin 6A significantly increases bacterial adherence to intact mouse corneas. The overall goal of this project is to understand mechanistic and functional aspects of KDAMP generation and antimicrobial activity. The hypothesis is that endogenous proteolytic processing of cytosolic keratin 6A generates keratin-derived antimicrobial peptides (KDAMPs), that keratin filament turnover enhanced by phosphorylation of its monomers increases generation and activity of KDAMPs, that KDAMPs kill bacteria by lysing bacterial membranes, and that KDAMPs protect the cornea against bacterial adherence. This will be tested in three aims. Aim 1 will determine how KDAMPs are generated by identifying the endogenous proteases involved and the role of phosphorylation-induced keratin solubilization in regulating levels and activity of KDAMPs. Aim 2 will explore the mechanisms by which KDAMPs kill bacteria by studying their structural interactions with bacterial membranes, and by identifying their bacterial receptors on Pseudomonas aeruginosa, a major corneal pathogen. Aim 3 will investigate the role of KDAMPs in protecting the cornea against P. aeruginosa. This will be done by localizing KDAMPs at the ocular surface, and determining their contribution to keratin 6A-mediated inhibition of bacterial adherence and subsequent corneal susceptibility to infection. Proteasome and protease inhibitors coupled with FPLC will be used to identify the proteases that generate KDAMPs. Phosphatase inhibitors, kinase activators and bacterial antigens will be used to promote keratin solubilization. Pore formation and peptide/membrane interactions will be investigated by liposomes and solid state NMR spectroscopy. Bacterial receptors will be identified by cellular fractionation and immunoprecipitation. Western blotting, ELISA and immunostaining with custom-made antibodies will be used to detect KDAMPs. Results of this study will improve our understanding of how the healthy cornea resists infection and could lead to novel therapeutic strategies for controlling infections of the cornea and other sites.