Abstract
The polyphenol (−)-epicatechin gallate (ECg) inserts into the cytoplasmic membrane (CM) of methicillin-resistant Staphylococcus aureus (MRSA) and reversibly abrogates resistance to β-lactam antibiotics. ECg elicits an increase in MRSA cell size and induces thickened cell walls. As ECg partially delocalizes penicillin-binding protein PBP2 from the septal division site, reduces PBP2 and PBP2a complexation and induces CM remodelling, we examined the impact of ECg membrane intercalation on phospholipid distribution across the CM and determined if ECg affects the equatorial, orthogonal mode of division. The major phospholipids of the staphylococcal CM, lysylphosphatidylglycerol (LPG), phosphatidylglycerol (PG), and cardiolipin (CL), were distributed in highly asymmetric fashion; 95%–97% of LPG was associated with the inner leaflet whereas PG (~90%) and CL (~80%) were found predominantly in the outer leaflet. ECg elicited small, significant changes in LPG distribution. Atomic force microscopy established that ECg-exposed cells divided in similar fashion to control bacteria, with a thickened band of encircling peptidoglycan representing the most recent plane of cell division, less distinct ribs indicative of previous sites of orthogonal division and concentric rings and “knobbles” representing stages of peptidoglycan remodelling during the cell cycle. Preservation of staphylococcal membrane lipid asymmetry and mode of division in sequential orthogonal planes appear key features of ECg-induced stress.
Highlights
IntroductionMembrane proteins that mediate essential functions such as signal transduction and protein secretion are laterally segregated into microdomains rich in lipid bilayer constituents, such as sphingolipids and sterols [1,2]
The heterogeneous nature of eukaryotic membranes is well established
To determine the impact of epicatechin gallate (ECg) on the molecular configuration of the cytoplasmic membrane (CM), we examined the distribution of LPG, PG and CL following intercalation of the polyphenol into the bilayer; we used atomic force microscopy to assess the effect of CM reconfiguration on the orthogonal mode of cell division [31]
Summary
Membrane proteins that mediate essential functions such as signal transduction and protein secretion are laterally segregated into microdomains rich in lipid bilayer constituents, such as sphingolipids and sterols [1,2]. The localization of protein complexes in these restricted membrane lipid rafts has profound importance for cell function [3] and cellular functions are strongly influenced by the different biophysical properties of the outer and inner surfaces of the membrane bilayer as a consequence of the non-random distribution of amphipathic components across the lipid palisade. Maintenance of the asymmetric distribution of phospholipids between the inner and outer leaflets of the plasma membrane is an energy-dependent process imposing a high metabolic burden on the cell [4,5]. Studies with lipid-binding fluorescent dyes have revealed that phospholipids may form microdomains within the cytoplasmic membrane (CM); for example, the poles and septa of
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