Cytoskeletal Keratins in Epithelial Immunity to Bacterial Keratitis

描述（由申请人提供）：细胞骨架角蛋白对细菌性角膜炎的上皮免疫角膜感染是全球成人和儿童视力损害和失明的主要原因。由于目前的治疗方法不足以预防视力丧失，因此需要其他治疗策略。在一项由盖茨基金会大挑战探索基金资助的研究中，我们探索了为什么角膜表面在清除接种在其表面的细菌方面非常有效。这项研究使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）相比，KDAMPs具有耐盐性，其杀伤作用不依赖于肽/细菌膜的静电吸引、肽疏水性和确定的二级结构，这表明它们与其他抗菌肽的不同之处在于它们如何与细菌膜相互作用。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粘附。该项目的总体目标是了解KDAMP产生和抗菌活性的机制和功能方面。该假说认为，胞质角蛋白6A的内源性蛋白水解过程产生角蛋白衍生的抗菌肽（KDAMPs），其单体磷酸化增强角蛋白丝的周转增加了KDAMPs的产生和活性，KDAMPs通过裂解细菌膜杀死细菌，并且KDAMPs保护角膜免受细菌粘附。这将在三个目标中得到检验。目的1将通过鉴定内源性蛋白酶以及磷酸化诱导的角蛋白溶解在调节水平和活性中的作用来确定KDAMPs是如何产生的

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.