Interaction of the Mammalian Antibacterial Peptide Cecropin P1 with Phospholipid Vesicles

Abstract

Cecropins are positively charged antibacterial polypeptides that were originally isolated from insects. Later on a mammalian homologue, cecropin P1 (CecP), was isolated from pig intestines. While insect cecropins are highly potent against both Gram-negative and Gram-positive bacteria, CecP is as active as insect cecropins against Gram-negative but has reduced activity against Gram-positive bacteria. To gain insight into the mechanism of action of CecP and the molecular basis of its antibacterial specificity, the peptide and its proline incorporated analogue (at the conserved position found in insect cecropins), P22-CecP, were synthesized and labeled on their N-terminal amino-acids with fluorescent probes, without significantly affecting their antibacterial activities. Fluorescence studies indicated that the N-terminal of CecP is located on the surface of phospholipid membranes. Binding experiments revealed that CecP binds acidic phosphatidylserine/phosphatidylcholine (PS/PC) vesicles better than zwitterionic PC vesicles, which correlates with its ability to permeate the former better than the latter. The shape of the binding isotherms suggest that CecP, like insect cecropin, binds phospholipids in a simple, noncooperative manner. However, resonance energy transfer (RET) measurements revealed that, unlike insect cecropins, CecP does not aggregate in the membrane even at relatively high peptide to lipid ratios. The stoichiometry of CecP binding to vesicles suggests that amount of CecP sufficient to form a monolayer causes vesicle permeation. In spite of the incorporation of the conserved proline in P22-CecP, the analogue has reduced antibacterial activity, which correlates with its reduced alpha-helical structure and its lower partitioning and membrane permeating activity with phospholipid vesicles. Taken together, our results support a mechanism in which CecP disrupts the structure of the bacterial membrane by (i) binding of peptide monomers to the acidic surface of the bacterial membrane and (ii) disintegrating the bacterial membrane by disrupting the lipid packing in the bilayers. These results, combined with data reported for other antibacterial polypeptides, suggest that the organization of peptide monomers within phospholipid membranes contributes to Gram-positive/Gram-negative antibacterial specificity.

Energetics of Pore Formation Induced by Membrane Active Peptides

“…We have argued, on the basis of the theory of membrane elasticity (66), that the peptide molecules embedded in the headgroup region are dispersed rather than aggregated. This was indeed supported by evidence from fluorescence energy transfer (67)(68)(69), NMR (58), and electron paramagnetic resonance (EPR, 70) studies. Thus the fractional increase of the monolayer area due to peptide…”

Interaction and Lipid-Induced Conformation of Two Cecropin−Melittin Hybrid Peptides Depend on Peptide and Membrane Composition

“…55 The initial phase in this model is, in fact, equivalent to the physical structure described by the "S" state in the two-state model by Huang. 22 As for Melittin, Yang et al 25 suggested that their results were compatible with the toroidal model for the interaction with zwitterionic membranes.…”

A Coiled-Coil Peptide Shaping Lipid Bilayers upon Fusion

“…The maxima of the emission l max were determined from adjacent average smoothed spectra. The data was interpreted as partition equilibrium (26)(27)(28)(29). The fluorescence intensities, F, in presence of vesicles were used to calculate the concentration of membrane-bound peptide, P b , by the following:…”