Bridging the Antimicrobial Activity of Two Lactoferricin Derivatives in E. coli and Lipid-Only Membranes

Abstract

We coupled the antimicrobial activity of two well-studied lactoferricin derivatives, LF11-215 and LF11-324, in Escherichia coli and different lipid-only mimics of its cytoplasmic membrane using a common thermodynamic framework for peptide partitioning. In particular, we combined an improved analysis of microdilution assays with ζ-potential measurements, which allowed us to discriminate between the maximum number of surface-adsorbed peptides and peptides fully partitioned into the bacteria. At the same time, we measured the partitioning of the peptides into vesicles composed of phosphatidylethanolamine (PE), phosphatidylgylcerol (PG), and cardiolipin (CL) mixtures using tryptophan fluorescence and determined their membrane activity using a dye leakage assay and small-angle X-ray scattering. We found that the vast majority of LF11-215 and LF11-324 readily enter inner bacterial compartments, whereas only 1−5% remain surface bound. We observed comparable membrane binding of both peptides in membrane mimics containing PE and different molar ratios of PG and CL. The peptides' activity caused a concentration-dependent dye leakage in all studied membrane mimics; however, it also led to the formation of large aggregates, part of which contained collapsed multibilayers with sandwiched peptides in the interstitial space between membranes. This effect was least pronounced in pure PG vesicles, requiring also the highest peptide concentration to induce membrane permeabilization. In PE-containing systems, we additionally observed an effective shielding of the fluorescent dyes from leakage even at highest peptide concentrations, suggesting a coupling of the peptide activity to vesicle fusion, being mediated by the intrinsic lipid curvatures of PE and CL. Our results thus show that LF11-215 and LF11-324 effectively target inner bacterial components, while the stored elastic stress makes membranes more vulnerable to peptide translocation.

Antimicrobial peptide activity in asymmetric bacterial membrane mimics

“…S2). Although increasing lipid concentration is known to affect peptide partitioning, 24,27 these data support the idea that L18W-PGLa does not preferentially partition into aLUVs under leakage conditions.…”

“…All three peptides are able to inhibit bacterial growth, with reported minimum inhibitory concentrations (MICs) of 16 µM (LF11-215), ∼ 31 µM (L18W-PGLa), and ∼ 62 µM (MG2a) for Escherichia coli K12. 14,24 Further, while the secondary structure of L18W-MG2a and MG2a once inserted into lipid membranes can be considered as mostly α-helical, 33,34 LF11-215 due to its short amino acid sequence is expected to be structurally more flexible. In fact, acylated LF11-215, with an octanoyl chain attached to the N-terminal, was reported to form a short α-helical-like turn of five residues in micelles.…”

“…Peptide partitioning was determined from Trp-fluorescence emission for L18W-PGLa and LF11-215 using a Cary Eclipse Fluorescence Spectrophotometer (Varian/Agilent Technologies) at a excitation wavelength of λ = 280 nm and slit widths for incident and outgoing beams of either 5 or 10 nm, as detailed previously. 24 All samples were contained in a quartz cuvette with a magnetic stirrer and measured at 37°C. Spectra were analyzed with a linear combination of two independent bands each fitted by a log-normal-like function.…”

“…25,26 This allowed us to extract the molar concentration of dissociated peptide, [P] W , and subsequently the molar concentration of membrane-associated peptide [P] B = [P] − [P] W , where [P] is the total peptide concentration in the sample. 24 The mole fraction partitioning coefficient…”

“…Specific care was taken to keep the peptide concentrations well below the thresholds reported for POPE/POPG (3:1 mol/mol) mixtures for the formation of vesicle aggregation or multilamellar vesicles. 24,31,32 This was additionally checked by inspection of the final SANS patterns after peptide addition, which did not show any signatures for changes in overall vesicle morphology or aggregation (Fig. S4).…”