In Vivo Efficacy of Keratin-Derived Antimicrobial Peptides (KDAMPs) in Corneal Defense Against Pseudomonas aeruginosa

Abstract

 

Purpose

 

We have shown that KDAMPs are novel antimicrobials constitutively expressed by corneal epithelial cells. Synthetic analogs of KDAMPs kill various bacterial pathogens of both Gram types in vitro. Specifically, they are rapidly bactericidal and cytoprotective against P. aeruginosa. In contrast to most known antimicrobial peptides, KDAMPs are unaffected by physiological salt concentration. As knockdown of keratin 6A significantly increases bacterial growth in human corneal epithelial cell lysates and bacterial adherence to intact mouse corneas, here we tested the hypothesis that exogenous addition of KDAMPs can protect corneas against P. aeruginosa in vivo.

 

Methods

 

Corneas of anesthetized C57BL/6 MyD88 knockout mice (known to be impaired in epithelial defense) were blotted with tissue paper to enhance susceptibility to bacterial adhesion, rinsed with physiological saline (0.9% NaCl), then inoculated with 10⁹ cfu in 5 ul GFP expressing P. aeruginosa PAO1. Eyes were rinsed with saline 2 h post-inoculation to remove non-adherent bacteria, followed by administration (once every 2 h) of 5 μl saline eye drops containing 200 μg/ml synthetic 19mer of KDAMPs or its scrambled control peptide. Animals were sacrificed 5 h post peptide treatment. Enucleated eyes were rinsed with PBS and unprocessed corneas/bacteria were imaged by confocal microscopy using 633 nm (red) and 488 nm (green) respectively. Z stacks images (entire corneal thickness, 1 μm steps) were collected from >10 random fields and reconstructed in 3-D by Image J.

 

Results

 

Blotted and inoculated mouse eyes instilled with scrambled 19mer control peptide or vehicle control (saline) in vivo were found to have massive P. aeruginosa colonization. In contrast, those instilled with synthetic 19mer KDAMP had significantly reduced bacterial adhesion on the cornea (Fig 1).

 

Conclusions

 

KDAMPs (i.e. the 19mer) are anti-pseudomonal not only in vitro but also in vivo. Further studies will be needed to understand the extent of their contributions to innate defense of the cornea, and if they can protect against infection once ongoing. Such studies could lead to novel therapeutic agents to prevent and/or treat infections of the cornea and other sites.

 

 

The 19mer KDAMP, but not scrambled control peptide, was effective in killing P. aeruginosa (green) colonized on the cornea (red) and protecting it against bacterial adhesion in vivo.

 

Keywords: 482 cornea: epithelium • 664 pseudomonas • 422 antibiotics/antifungals/antiparasitics