Antimicrobial peptides are a potent class of antibiotics. In the Gram-positive model organism Bacillus subtilis the synthetic peptide RWRWRW-NH2 integrates into the bacterial membrane and delocalizes essential peripheral membrane proteins involved in cell wall biosynthesis and respiration. A lysine residue has been added to the hexapeptide core structure, either C or N-terminally. Lipidation of the lysine residues by a C8-acyl chain significantly improved antibacterial activity against both Gram-positive and Gram-negative bacteria. Here, we report a comparative proteomic study in B. subtilis on the mechanism of action of the lipidated and non-lipidated peptides. All derivatives depolarized the bacterial membrane without forming pores and all affected cell wall integrity. Proteomic profiling of the bacterial stress responses to the small RW-rich antimicrobial peptides was reflective of non-disruptive membrane integration. Overall, our results indicate that antimicrobial peptides can be derivatized with lipid chains enhancing antibacterial activity without significantly altering the mechanism of action. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

The attachment of lipids to C- or N-terminally positioned lysine side-chain amino groups increases the activity of a short synthetic (Arg-Trp)(3) antimicrobial peptide significantly, making these peptides even active against pathogenic Gram-negative bacteria. Thus, a peptide with strong activity against S. aureus (1.1-2 mu M) and good activity against A. baumannii and P. aeruginosa (9-18 mu M) was identified. The most promising peptide causes 50% hemolysis at 285 mu M and shows some selectivity against human cancer cell lines. Interestingly, the increased activity of ferrocenoylated peptides is mostly due to the lipophilicity of the organometallic fragment.