Bacterial keratitis is a major cause of ocular surface morbidity that often leads to permanently compromised visual function, sometimes to an extent that necessitates corneal transplantation to restore adequate vision. Often the bacterial strains that cause these infections are resistant to treatment with traditional antibiotics. Due to their role in innate immunity at the ocular surface, antimicrobial peptides (AMPs) show promise as a new generation of antibiotics against resistant strains. However, single AMPs can be toxic to host cells at the concentrations necessary to have the same microbicidal efficacy as traditional antibiotics, a fact which has impeded progress in the development of AMPs as therapeutic agents for the ocular surface. The current knowledge base suggests that effective microbicidal activity of AMPs is achieved in vivo without significant host cell toxicity due to a combination of high local concentration, multimericity and synergism. Our objectives are to understand how the spatial arrangement, multimeric state, and synergism of AMPs present at the ocular surface enhance microbicidal activity and prevent toxicity to corneal epithelial cells. We expect that 1) multimers of a single AMP in highly localized micro-domains will overcome the threshold concentration necessary to confer strong microbicidal properties while remaining non-toxic to the corneal epithelium and 2) synergy of AMPs in micro-domains will further reduce both corneal epithelial cell cytotoxicity and the effective concentration of each AMP necessary to confer microbicidal activity. We will test our hypotheses by immobilizing AMPs in varying densities, ratios and patterns onto biocompatible surfaces and assessing the toxicity to bacteria and corneal epithelial cells. Our approach using conducting AFM and film deposition to pattern AMPs is innovative because it is based on state-of-the-art nanofabrication techniques to address questions about innate immunity of the ocular surface. Upon completion of our proposed studies, we expect to understand how multimeric state and synergism relate to microbicidal activity. Establishing this relationship will increase our understanding of innate immunity at the ocular surface and further the development of AMPs as effective antimicrobial agents. As clinically relevant pathogens become resistant to available antibiotics, new methods to combat infection must be developed. Antimicrobial peptides of the innate immune system present an alternative because they are derived from host tissue and are not likely to lead to resistance. We propose to increase the understanding of how these peptides function so that they can be developed as a new class of antimicrobial compounds.
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