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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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 5

The K6A-derived 19-mer binds to bacterial cells and permeabilizes their cell membranes.

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The K6A-derived 19-mer binds to bacterial cells and permeabilizes their ...
(A) P. aeruginosa strain 6206 was either untreated (control) or treated with an equimolar concentration (110 μM each) of scrambled 19-mer (200 μg/ml), TAMRA (50 μg/ml), or 19-mer (200 μg/ml) for 3 hours and then stained with a cell-impermeant dye SYTOX Blue. Only the 19-mer–treated bacteria were stained with SYTOX Blue. TAMRA itself also did not bind to bacteria. Original magnification, ×1,000. BF, bright field. (B) Strain 6206 was incubated with TAMRA-tagged (N-terminal) 19-mer (110 μM, equivalent to 250 μg/ml) for 3 hours then stained with SYTOX Blue. Fluorescence microscopy showed that the bacteria were labeled with the TAMRA-tagged 19-mer peptide (red) and SYTOX (blue) and that these labels colocalized. Original magnification, ×1,000. Higher-magnification images are shown in inset panels (original magnification, ×~2,000). (C) TAMRA-tagged 19-mer had similar bactericidal activity to that of unlabeled peptide (93.2% and 98.4%, respectively, P < 0.05) after 3 hours at 110 μM against P. aeruginosa (strain 6206). TAMRA alone was inactive. (D) Rapid and extensive killing action of the 19-mer against P. aeruginosa strain 6294 and strain 6206. (E) SYTOX staining of P. aeruginosa 6206 at 5 and 15 minutes after treatment with the 19-mer KDAMP at 75 μg/ml. Bacteria incubated with the scrambled 19-mer or untreated served as controls. DIC, differential interference contrast. Original magnification, ×400.