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G. Qin, C. Cai, A. McDermott; Influence of Local Concentration on the Antimicrobial Efficacy of the Cathelicidin LL-37. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4790.
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LL-37 is an antimicrobial peptide expressed by ocular surface epithelial cells that has antibacterial activity and can modulate immune responses and wound healing and so has potential clinical uses. Evidence suggests that a high local concentration of antimicrobial peptides present in vivo facilitates the disruption of bacterial membranes. Here we investigated the effect of localizing LL-37 to surfaces on its antibacterial activity.
LL-37 was immobilized onto oligo (ethylene glycol) monolayers presenting carboxylic acid groups on gold substrates through electrostatic interactions. The coated surfaces were characterized by X-ray photoelectron spectroscopy and ellipsometry. The antibacterial effect of immobilized LL-37 was measured against a P. aeruginosa strain expressing green fluorescent protein (PA01-GFP). The coated surfaces were incubated with 100ul of a 4x108 cfu/ml bacterial suspension for up to 2 h. Propidium iodide was used to stain bacteria with compromised cell membranes. Fluorescence images were captured digitally and the number of bacteria was counted using ImagePro software.
Analysis showed that the surface concentration of LL-37 was on the order of 1013 molecules/cm2, equivalent to a concentration in the ng/mL range were the peptide to be dissolved in solution under the current conditions. Previous studies showed that effective killing by LL-37 in solution required concentrations in the ug/ml range. At a surface concentration of 4.4x1013 molecules/cm2 LL-37 showed significant antimicrobial efficacy with ~ 97% of PA01-GFP adsorbed on the surfaces having compromised membranes (n=3). Notably, we observed a sharp increase in bacterial killing when the surface density of LL-37 exceeded 3.3x1013 molecules/cm2, suggesting cooperative effects. Furthermore, a kinetic study showed that the adsorption and killing of bacterial cells occurred within 3 min.
Our findings suggest that localization of LL-37 on surfaces greatly increased its antimicrobial efficacy, that molecules of LL-37 act cooperatively and that the adsorption and killing of bacterial cells were rather rapid. These results indicate that LL-37 may have significant activity in vivo when presented in an appropriate manner thus may be useful clinically to prevent and treat ocular surface infection.
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