Cytoskeletal Keratins in Epithelial Immunity to Bacterial Keratitis

描述（由申请方提供）：细胞骨架角蛋白在细菌性角膜炎上皮免疫中的作用角膜感染是全世界成人和儿童视力受损和失明的主要原因。由于目前的治疗不足以预防视力丧失，因此需要替代治疗策略。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上的细菌方面非常有效。这项研究使我们发现了一类新型的抗菌肽（KDAMP），它们是角蛋白6A的富含甘氨酸的C-末端片段，角蛋白6A是一种在广泛上皮细胞的细胞骨架中发现的主要中间丝蛋白。与大多数已知的抗菌肽（AMP）相比，KDAMP是耐盐的，并且它们的杀伤作用不依赖于肽/细菌膜静电吸引、肽疏水性和限定的二级结构，这表明它们与其他AMP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，敲低角蛋白6A显著增加细菌对完整小鼠角膜的粘附。该项目的总体目标是了解KDAMP生成和抗菌活性的机制和功能方面。假设是细胞溶质角蛋白6A的内源性蛋白水解加工产生角蛋白衍生的抗微生物肽（KDAMP），通过其单体的磷酸化增强的角蛋白丝周转增加KDAMP的产生和活性，KDAMP通过溶解细菌膜杀死细菌，并且KDAMP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将通过鉴定参与的内源性蛋白酶以及磷酸化诱导的角蛋白溶解在调节KDAMPs的水平和活性中的作用来确定KDAMPs是如何产生的。 KDAMP。目的2将通过研究KDAMPs与细菌膜的结构相互作用，并通过鉴定其在铜绿假单胞菌（一种主要的角膜病原体）上的细菌受体来探索KDAMPs杀死细菌的机制。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。这将通过将KDAMP定位在眼表，并确定其对角蛋白6A介导的细菌粘附抑制和随后的角膜感染易感性的贡献来完成。将使用与FPLC偶联的蛋白酶体和蛋白酶抑制剂来鉴定产生KDAMP的蛋白酶。磷酸酶抑制剂、激酶激活剂和细菌抗原将用于促进角蛋白溶解。将通过脂质体和固态NMR光谱研究孔形成和肽/膜相互作用。细菌受体将通过细胞分级分离和免疫沉淀来鉴定。将使用Western印迹法、ELISA法和使用定制抗体的免疫染色法来检测KDAMP。这项研究的结果将提高我们对健康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.