Selectivity of Antimicrobial Peptides: Association to Bacterial and Eukaryotic Cells and Cell-Density Dependence

Abstract

Several questions limit the applicability of antimicrobial peptides (AMPs) as a new class of antibiotics against the problem of multidrug resistant bacteria.AMPs are screened for their direct in vitro bactericidal activity, mediated by association to and perturbation of bacterial membranes. However, they act also as immunomodulators, and it is debated which function is predominant at peptide and bacteria concentrations relevant for in vivo conditions.In vitro, AMPs are toxic to mammalian cells only at concentrations higher than those needed for bactericidal activity. This selectivity is presumably determined by the difference in lipid composition of membranes of the two cell types, as studies on liposomes show a higher affinity for bilayers mimicking bacterial membranes. However, it might be just an experimental artifact of the in vitro assay conditions that employ lower cell densities for bacteria than for red blood cells (RBCs), which are also larger.Recently, by fluorescence spectroscopy we characterized the association of the cathelicidin PMAP-23 to bacterial cells, and determined for the first time the number of membrane-bound peptide molecules necessary to kill a bacterial cell (106 peptides per E. Coli cell) [ACS Chem. Biol. 2014 9:2003]. We now measured the dependence of killing activity on bacterial cell density: as a consequence of the binding equilibrium, total micromolar peptide concentrations are needed even with extremely low bacterial counts. We also extended our method to measure peptide binding to RBCs, showing that also in this case a high coverage of the cell surface is needed to cause lysis. Finally, we investigated the cell-density dependence of hemolytic activity.Overall, these data clarify the complex dependence of AMP activity and selectivity on the density of bacterial and human cells, and open new questions for their behavior in vivo.