Model membrane approaches to determine the role of calcium for the antimicrobial activity of friulimicin

Friulimicin is a cyclic lipopeptide antibiotic, currently in clinical development, that possesses excellent activity against Gram-positive bacteria, including multiresistant strains. A recent study on the mode of action of friulimicin reported on the interference with bacterial cell wall biosynthesis via a calcium-dependent complexing of the bactoprenol phosphate carrier C₅₅-P. The calcium dependency of this non-common targeted activity remains to be elucidated. In the present model membrane approach, the role of calcium for friulimicin targeting to C₅₅-P was investigated by biosensor-based detection of binding affinities. The findings were supplemented by atomic force microscopy (AFM) and circular dichroism (CD) spectroscopy. Comparing the calcium salt of friulimicin with the calcium-free peptide, calcium appeared to be essential for friulimicin interaction with DOPC model membranes. The binding affinity was even higher in the presence of 0.1 mol% C₅₅-P (0.21 μM vs. 1.22 μM), confirming the targeted mode of action. Binding experiments with supplemented calcium salts suggest (i) the phosphate group as the essential moiety of C₅₅-P, referring to a bridging function of calcium between the negatively charged friulimicin and C₅₅-P, and (ii) a structural effect of calcium shifting the peptide into a suitable binding conformation (CD spectra). AFM images confirmed that calcium has no, or only a minor, effect on the aggregate formation of friulimicin. These data shed new light on the mechanisms of antibacterial activity of friulimicin.