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Connie Tam, James J. Mun, David J. Evans, Suzanne M. Fleiszig; Efficacy and Structure-Activity of Keratin-Derived Antimicrobial Peptides (kDAMPs): A Novel Role for Intermediate Filament Proteins in Corneal Innate Defense. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3146.
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© ARVO (1962-2015); The Authors (2016-present)
Antimicrobial peptides (AMPs) expressed by corneal epithelial cells are thought to contribute to corneal innate defense. We have previously identified a novel family of constitutively-expressed AMPs derived from the glycine-rich C-terminus of keratin 6A, an intermediate filament protein of mammalian cells. Synthetic analogs of kDAMPs (10 to 36 amino acids) possess potent antibacterial and cytoprotective activity against Pseudomonas aeruginosa. Here we examined the structure and activity spectra of kDAMPs to gain insights into their mode of action.
CD spectroscopy and the K2D2 program were used to analyze secondary structures of synthetic kDAMPs in pure water, a pure water/trifluoroethanol (TFE) mixture, or pure TFE. Antimicrobial activity was determined against P. aeruginosa clinical isolates (6206 and 6294), or ATCC strains of E. coli, Serratia marcescens, Staphylococcus epidermidis, S. aureus and Streptococcus pyogenes using viable counts after exposure to 200 μg/ml of peptide. K6A was knocked-down in wild-type C57BL/6 mice via subconjunctival injection of siRNA.
kDAMPs were rapidly anti-Pseudomonal (except scrambled controls), e.g. after 15 min, a 19mer killed 92% of the cytotoxic strain 6206, and 98% of the invasive strain 6294 (P<0.05). Moreover, knockdown of keratin 6A significantly increased P. aeruginosa adherence to the intact corneal surface ex vivo. kDAMPs were active against other bacteria. After 3 h, the 19mer, 18mer (lacking a N-terminal residue) and a 14mer all killed S. pyogenes (>99.9%, P<0.0001). The 14mer also killed S. epidermidis (>99.8%, P <0.0001) and S. aureus (97%, P<0.0005). The 18mer and 14mer killed E. coli (98%, >99.9%, P <0.001 respectively), and a 36mer killed S. marcesens (40%, P<0.05). All kDAMPs tested, including the inactive 19mer, had negative ellipticity values in water and 50% TFE at the 200 nm region suggesting random coil structure. In pure TFE, kDAMPs remained unstructured except the 36mer. K2D2 analysis confirmed peptides with random coil spectra had negligible helical content. However, glycine to alanine substitution (G2A/G8A) in a 10mer reduced activity versus P. aeruginosa by 10-fold.
kDAMPs are glycine-rich, non-helical, highly flexible antimicrobial peptides with broad efficacy against multiple bacterial pathogens, and contribute to corneal resistance to bacterial adherence. Further studies are needed to determine mechanism(s) of kDAMP activity, and if site-specific proteolytic degradation of keratins during intermediate filament turnover provides a novel innate defense mechanism for the cornea, and other mucosal epithelial barriers.
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