Abstract
Purpose :
The cationic antimicrobial protein of 37kDa (CAP37) is constitutively expressed in human neutrophils and plays a significant role in host defense against infection due to its potent bactericidal activity for Gram-negative bacteria. Our goal is to develop therapeutic peptides derived from CAP37 for the treatment of vision-threatening corneal infections.
Methods :
The primary antibiotic domain of CAP37 resides between amino acid 20 and 44 of the native protein. We synthesized a peptide that included a modification of the native sequence by changing the cysteine residue at position 26 to a serine, the addition of one arginine (R) residue at the carboxy terminus, and 4 R residues and 2 mini-pegs (MP) at the amino terminus. The resulting peptide 20-44ser26-5RMP demonstrated enhanced bactericidal activity for Gram negative pathogens. Antibacterial activity, calculated as a reduction in colony forming units, was determined in vitro and in vivo using a mouse model of bacterial keratitis. Cytotoxicity was determined by red blood cell hemolysis and by assessing cytokine production in vivo. Lipopolysaccharide (LPS) binding activity was determined using the chromogenic limulus amebocyte lysate assay. Binding to toll-like receptor 4 (TLR4) was determined by enzyme-linked immunosorbent assay, and activation was quantified in HEK-blueTM hTLR4 cells.
Results :
In vitro bactericidal assays demonstrated that the peptide killed Gram-negative clinical isolates with multi-drug resistance profiles (Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli) at 25 μg/ml. A topical dose of 250 μg/ml cleared the bacterial infection induced by P. aeruginosa in a mouse keratitis model. The peptide binds and neutralizes the toxic effects of LPS. It binds but does not activate TLR4, at concentrations up to 400 μg/ml. Activation of TLR4 by the agonist S100A9 was inhibited by the peptide. The peptide has low cytotoxicity (<1% hemolysis), at concentrations up to 500 μg/ml and does not induce a cytokine storm when administered intravenously.
Conclusions :
In conclusion, CAP37-derived peptide 20-44ser26-5RMP has low toxicity, is efficient in killing multi-drug resistant Gram-negative bacteria, has LPS-neutralizing activity and can inhibit TLR4 activation, which makes it a strong candidate for use as a therapeutic for serious Gram-negative keratitis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.