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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

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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 1

Keratin peptide derivatives are major constituents of the antimicrobial fractions of human corneal epithelial cell lysates.

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Keratin peptide derivatives are major constituents of the antimicrobial ...
(A) Crude lysates were serially fractionated by size to yield 4 fractions: >100 kDa, 10–100 kDa, 3–10 kDa, and <3 kDa (black). Crude lysates and the small-molecule <3-kDa lysate fraction showed bactericidal activity against P. aeruginosa (strain PAC1R). Culture media was fractionated in parallel to serve as controls for the corresponding lysate fractions in the bacterial killing assays (white). (B and C) Similar results were observed for P. aeruginosa clinical isolate (B) 6294 (invasive strain) and (C) 6206 (cytotoxic strain) except that 3- to 10-kDa lysate fractions were also found to be bactericidal. Dilution (1:3) (gray) reduced activity, demonstrating that antimicrobial activity was concentration dependent. (D) The <3-kDa and 3- to 10-kDa fractions both protected epithelial cells against P. aeruginosa internalization. (E) The <3-kDa fraction protected epithelia from bacterial-induced cell death (indicated by trypan blue–staining).

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ISSN: 0021-9738 (print), 1558-8238 (online)

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