Lipid domain formation and non-lamellar structures associated with varied lysylphosphatidylglycerol analogue content in a model Staphylococcal plasma membrane

Reg Rehal,Robert D Barker,Zidong Lu,Tam T Bui,Bruno Demé,Gerd Hause,Christian Wölk,Richard D Harvey

doi:10.1016/j.bbamem.2021.183571

Abstract

Dipalmitoyl-3-aza-dehydroxy-lysylphosphatidylglycerol (DP3adLPG), is a chemically stable synthetic analogue of the bacterial lipid lysylphosphatidylglycerol (LPG), designed as a substitute for the notoriously labile native lipid in biophysical investigations. In Staphylococcus aureus, LPG is known to play a role in resistance to antibiotics by altering membrane charge properties in response to environmental stress, but little is known about how LPG influences other bilayer physicochemical properties or lateral organisation, through the formation of complexes with lipids such as phosphatidylglycerol (PG). In this study we have investigated the different phases formed by biomimetic mixtures of 3adLPG and PG in different thermotropic states, using neutron diffraction and electron microscopy. In a DPPG/DP3adLPG 70:30 mol% mixture, two distinct lamellar phases were observed below the lipid melting transition: Lβ′ 1 and Lβ′ 2 with respective periodicities of 82 and 62 Å. Increasing the proportion of DP3adLPG to mimic the effects of environmental stress led to the disappearance of the Lβ′ 1 phase and the formation of an inverse hexagonal phase. The compositions of these different phases were identified by investigating the thermotropic properties of the two mixtures, and probing their interaction with the antimicrobial peptide magainin 2 F5W. We propose that the observed polymorphism results from the preferential formation of either triplet PG-3adLPG-PG, or paired PG-3adLPG complexes, dependent upon the mixing proportions of the two lipids. The relevance of these findings to the role native LPG in S. aureus, are discussed with respect to their influence on antibiotic resistance and lateral membrane organisation.

Highlights

The role played by the aminoacyl lipid lysylphosphatidylglycerol (LPG) (Fig. 1A) in resistance to host defensive cationic antimicrobial peptides (CAPs) in Staphylococcus aureus and other Gram positive bac teria, has been elucidated mostly through in vitro genotypic and phenotypic studies
In a parallel study of peptide binding to the lipid dispersions, using the fluorescence emission of the tryptophan residue as a reporter (Fig. 2B), the reduction in helical content of the peptide coincides with a reduction in detectable binding to the lipids (Table 2). These results are similar to those we have previously reported for magainin 2 F5W interactions with DP3adLPG-containing vesicles at 55 ◦C, where higher DPPG content resulted in high amounts of α-helix, which are reduced almost to zero in near equimolar anionic/
As the samples prepared for the neutron diffraction measurements were solvated by water or D2O vapour, the pH of the solvent layers would be determined by the concentrations of the two lipid counter ions, triethylammonium (d62DPPG) and trifluoroacetate (d62DP3adLPG)

Summary

Introduction

The role played by the aminoacyl lipid lysylphosphatidylglycerol (LPG) (Fig. 1A) in resistance to host defensive cationic antimicrobial peptides (CAPs) in Staphylococcus aureus and other Gram positive bac teria, has been elucidated mostly through in vitro genotypic and phenotypic studies. Since the proportion of LPG present in the membrane outer leaflet has rarely been shown to exceed 30% of the total phospholipids, even in resistant phenotypes [2,3,7,8,9], it must be assumed to be fully seques tered into ion pairs by the more abundant PG, contributing to an overall charge dampening of the plasma membrane [10], when sym metrically distributed between the bilayer leaflets [3] In this more nuanced picture of the role of LPG in membrane defence, it is increased neutralisation of PG which facilitates CAP resistance and not the cu mulative effect of discrete regions of charge inversion

Methods

Results

Conclusion