Cytokeratins mediate epithelial innate defense through their antimicrobial properties
Connie Tam, … , David J. Evans, Suzanne M.J. Fleiszig
Connie Tam, … , David J. Evans, Suzanne M.J. Fleiszig
Published September 24, 2012
Citation Information: J Clin Invest. 2012;122(10):3665-3677. https://doi.org/10.1172/JCI64416.
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Research Article

Cytokeratins mediate epithelial innate defense through their antimicrobial properties

Abstract

Epithelial cells express antimicrobial proteins in response to invading pathogens, although little is known regarding epithelial defense mechanisms during healthy conditions. Here we report that epithelial cytokeratins have innate defense properties because they constitutively produce cytoprotective antimicrobial peptides. Glycine-rich C-terminal fragments derived from human cytokeratin 6A were identified in bactericidal lysate fractions of human corneal epithelial cells. Structural analysis revealed that these keratin-derived antimicrobial peptides (KDAMPs) exhibited coil structures with low α-helical content. Synthetic analogs of these KDAMPS showed rapid bactericidal activity against multiple pathogens and protected epithelial cells against bacterial virulence mechanisms, while a scrambled peptide showed no bactericidal activity. However, the bactericidal activity of a specific KDAMP was somewhat reduced by glycine-alanine substitutions. KDAMP activity involved bacterial binding and permeabilization, but the activity was unaffected by peptide charge or physiological salt concentration. Knockdown of cytokeratin 6A markedly reduced the bactericidal activity of epithelial cell lysates in vitro and increased the susceptibility of murine corneas to bacterial adherence in vivo. These data suggest that epithelial cytokeratins function as endogenous antimicrobial peptides in the host defense against infection and that keratin-derived antimicrobials may serve as effective therapeutic agents.

Authors

Connie Tam, James J. Mun, David J. Evans, Suzanne M.J. Fleiszig

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Figure 3

Synthetic analogs of K6A-derived peptides found in corneal epithelial cell lysates are bactericidal and cytoprotective against P. aeruginosa.

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Synthetic analogs of K6A-derived peptides found in corneal epithelial ce...
(A) The 19-mer was tested against P. aeruginosa strain 6206 (clinical isolate) in physiological saline (0.9% NaCl) or water and was found to be salt-tolerant (*P < 0.004, versus no peptide control). (B) The 19-mer peptide showed dose-dependent bactericidal activity against resuspended lawn culture of P. aeruginosa invasive strains 6294 (clinical isolate) and PAO1 (laboratory strain), with optimal activity at 200 μg/ml under high-salt conditions. (C) At 200 μg/ml, a scrambled control of the 19-mer peptide was inactive against P. aeruginosa. (D) Invasion of epithelial cells (strain 6294) (*P = 0.0008, versus no peptide control) and (E) cytotoxicity toward epithelial cells (strain 6206) were prevented by the 19-mer K6A-derived peptide (*P = 0.0009, versus no peptide control). (F) Synthetic variants of the 19-mer also showed dose-dependent bactericidal activity against P. aeruginosa strain 6206 (clinical isolate). Peptide as small as 10–amino acids retained bactericidal activity, despite a predicted loss of cationic charge (at pH 7.0), hydrophobic face, and ability to form a transmembrane helix (e.g., 13-mer, 10-mer). (G) Bactericidal activity of 19-mer (200 μg/ml) was only slightly affected by mutations in LPS for P. aeruginosa (strain PAC1R, serotype 03). PAC1RalgC- and PAC605 have incomplete LPS core oligosaccharide and are O antigen deficient (27), and PAC557 has a complete core but is O antigen deficient, while PAC611 has mutations only in the core (64).