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.
Corneal infection remains a major course of blindness. This proposal expands upon our discovery of a novel class of antimicrobial peptides in corneal cells (KDAMPs) by furthering our understanding of their generation, regulation, bacterial killing mechanisms, and roles in inhibiting bacterial adhesion to the cornea. This study could lead to new therapeutic agents or strategies for prevention and treatment of corneal and other infections.
