Evidence for a Novel Mechanism of Antimicrobial Action of a Cyclic R-,W-Rich Hexapeptide

Abstract

The development of antimicrobial peptides as new class of antibiotic agents requires structural characterisation and understanding of their diverse mechanisms of action. As the cyclic hexapeptide cWFW (cyclo(RRRWFW)) does not exert its rapid cell killing activity by membrane permeabilisation, in this study we investigated alternative mechanisms of action, such as peptide translocation into the cytoplasm and peptide interaction with components of the phospholipid matrix of the bacterial membrane. Using fluorescence microscopy and an HPLC-based strategy to analyse peptide uptake into the cells we could confirm the cytoplasmic membrane as the major peptide target. However, unexpectedly we observed accumulation of cWFW at distinct sites of the membrane. Further characterisation of peptide-membrane interaction involved live cell imaging to visualise the distribution of the lipid cardiolipin (CL) and isothermal titration calorimetry to determine the binding affinity to model membranes with different bacterial lipid compositions. Our results demonstrate a distribution of the cyclic peptide similar to that of cardiolipin within the membrane and highly preferred affinity of cWFW for CL-rich phosphatidylethanolamine (POPE) matrices. These observations point to a novel mechanism of antimicrobial killing for the cyclic hexapeptide cWFW which is neither based on membrane permeabilisation nor translocation into the cytoplasm but rather on preferred partitioning into particular lipid domains. As the phospholipids POPE/CL play a key role in the dynamic organisation of bacterial membranes we discuss the consequences of this peptide-lipid-interaction and outline the impact on antimicrobial peptide research.

Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis

“…In agreement with the cytoplasmic membrane as the likely cellular target6, cWFW elicited a typical cell envelope stress response characterised by upregulation of proteins controlled by the extracytoplasmic function (ECF) sigma factors SigM, SigW and SigX (Table 1). In addition, marker proteins indicative of membrane damage (phage shock protein PspA and NAD synthase NadE) and impairment of membrane-bound steps of cell wall synthesis (PspA homologue LiaH and the Sigma M-controlled protein YpuA) were upregulated.…”

“…Earlier investigations have suggested that cWFW does not permeabilize the cytoplasmic membrane of E. coli , or form pores in model membranes56. However, the induction of the general SigB-dependent stress response and the similarity of the proteomic response triggered by MP196 and gramicidin S suggests that cWFW might nevertheless cause energy starvation linked to membrane depolarisation in B. subtilis .…”

“…In contrast to most AMPs, however, cWFW does not permeabilize the cell membrane to large molecules56. As first postulated by Epand and Epand7, short cationic AMPs might, as an alternative mechanism, induce clustering of anionic lipids within the bacterial membrane due to electrostatic interactions8.…”

Lights, Camera, Action! Antimicrobial Peptide Mechanisms Imaged in Space and Time

“…The Dathe lab has carried out a series of studies of cyclic hexapeptide interactions with single, live E. coli and B. subtilis cells [24]. These peptides are attractive drug candidates due to their resistance to proteolysis.…”

“…In early work, a standard assay using the β-galactosidase substrate ONPG showed that lethal dosages of c -WFW never permeabilize the CM of E. coli . A recent imaging study using an analogue labeled with the fluorescent dye NBD (4-chloro-7-nitrobenzofurazan), called c -W(NBD)W (Figure 3) found strong accumulation of the peptide at the CM and no evidence that the peptide ever accesses the bulk of the cytoplasm [24]. In B. subtilis , both c -W(NBD)W (Figure 3) and the CL stain NAO showed similar patterns, a thin staining of the CM plus strong accumulation at the septal and polar regions.…”