Molecular Transport and Growth of Lipid Vesicles Exposed to Antimicrobial Peptides.

Josefine Eilsø Nielsen,Reidar Lund

doi:10.1021/acs.langmuir.1c02736

Josefine Eilsø Nielsen, Reidar Lund

Open Access

https://doi.org/10.1021/acs.langmuir.1c02736

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Abstract

It is well-known that lipids constituting the cytoplasmic membrane undergo continuous reorganization to maintain the appropriate composition important for the integrity of the cell. The transport of lipids is controlled by mainly membrane proteins, but also spontaneous lipid transport between leaflets, lipid “flip–flop”, occurs. These processes do not only occur spontaneously under equilibrium, but also promote structural rearrangements, morphological transitions, and growth processes. It has previously been shown that intravesicular lipid “flip–flop” and intervesicular lipid exchange under equilibrium can be deduced indirectly from contrast variation time-resolved small-angle neutron scattering (TR-SANS) where the molecules are “tagged” using hydrogen/deuterium (H/D) substitution. In this work, we show that this technique can be extended to simultaneously detect changes in the growth and the lipid “flip–flop” and exchange rates induced by a peptide additive on lipid vesicles consisting of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), d-DMPC (1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine), DMPG (1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), and small amounts of DMPE-PEG (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). Changes in the overall size were independently monitored using dynamic light scattering (DLS). We find that the antimicrobial peptide, indolicidin, accelerates lipid transport and additionally induces limited vesicular growth. Moreover, in TR-SANS experiments using partially labeled lipid mixtures to separately study the kinetics of the lipid components, we show that, whereas peptide addition affects both lipids similarly, DMPG exhibits faster kinetics. We find that vesicular growth is mainly associated with peptide-mediated lipid reorganization that only slightly affects the overall exchange kinetics. This is confirmed by a TR-SANS experiment of vesicles preincubated with peptide showing that after pre-equilibration the kinetics are only slightly slower.

Highlights

The cell membrane relies on controlled transport through the membrane to maintain its integrity, because an exact composition in terms of lipid and ions is required for healthy cell function
We investigate peptide-induced vesicular growth as well as lipid exchange and “flip−flop” dynamics using the kinetic zero-average contrast (KZAC) time-resolved small-angle neutron scattering (TR-SANS) method combined with dynamic light scattering (DLS)
The TR-SANS method illustrated in Figure 2 is based on mixing protiated, H-labeled and deuterated, D-labeled vesicles and observing the decay in the scattering intensity over time

Summary

Introduction

The cell membrane relies on controlled transport through the membrane to maintain its integrity, because an exact composition in terms of lipid and ions (protons, sodium, calcium, etc.) is required for healthy cell function. The balance is mainly kept by transmembrane proteins, which accurately regulate the composition of lipids and the balance of ions.[1] The cytoplasmic membrane of eukaryotic and prokaryotic cells requires maintaining an asymmetric lipid composition on both the inner and the outer leaflets to function. In contrast to inplane diffusion, it has long been known that lipid “flip−flop” is relatively slow (minutes−hours−days−months) in the absence of transmembrane proteins (“scramblases”, “flippases” and “floppases”),[2] which have been found to significantly accelerate the process (seconds).[1,3,4] Flippases and floppases are adenosine triphosphate (ATP)-dependent membrane proteins, as opposed to ATP-independent scramblases, which all move lipids to the inner monolayer and outer monolayer, respectively,[5] and in that manner carefully maintain the lipid composition and rejuvenate the outer leaflet as lipids are synthesized within the cytoplasm. Destabilization of the bacterial membrane through accelerated lipid “flip−flop” has further been suggested as an essential step in the mode of action of antimicrobial peptides (AMPs).[6−13]

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