Binding of Daptomycin to Anionic Lipid Vesicles Is Reduced in the Presence of Lysyl-Phosphatidylglycerol

Abstract

The cytoplasmic membrane of Staphylococcus aureus contains ϳ20 mol% of the net cationic lipid lysyl-phosphatidylglycerol (LPG). Elevated fractions of LPG are associated with increased resistance to cationic antibiotics, including the lipopeptide daptomycin (DAP). Although the surface charge of the bacterial cytoplasmic membrane is altered by LPG, surface binding of DAP was found to be only moderately affected in anionic vesicles containing 20 mol% LPG. These results suggest that charge repulsion cannot fully explain LPG-mediated resistance to cationic peptides. L ysylated phosphatidylglycerol (LPG) is a common aminoacylated phospholipid in the cytoplasmic membranes (CMs) of Staphylococcus aureus, where it constitutes about 20 mol% of the total CM phospholipid fraction (1-4). Phosphatidylglycerol (PG) is the most abundant phospholipid in S. aureus, imparting an overall negative surface charge to the bacterial CM. In LPG, lysine is esterified to the glycerol headgroup of PG, effectively replacing an anionic lipid with one that carries a net cationic charge. Elevated levels of LPG correlate with resistance to killing by the cationic, calcium-dependent lipopeptide daptomycin (DAP), as well as cationic host defense peptides in S. aureus and Listeria spp.(5-12). The effect of LPG on the efficacy of cationic antimicrobial peptides has been attributed to the reduced negative surface charge of the cytoplasmic membrane as a consequence of the aminoacylation of PG to LPG (13). This interpretation was challenged in a liposomal model system in which LPG levels typically found in clinical S. aureus strains only minimally affected binding of the alpha-helical antimicrobial peptides RP-1, cecropin A, and mastoparan X (14, 15). In those cases, the protective effect of lysylated phospholipids against antimicrobial peptides was attributed to a stabilization of the bilayer in the presence of lysylated phospholipids, leading to significantly reduced peptide-induced CM perturbations.RP-1, cecropin A, and mastoparan X belong to a class of short, linear cationic peptides that adopt an alpha-helical conformation when bound to lipid membranes. DAP, like RP-1, cecropin A, and mastoparan X, binds with preference to the anionic CMs typically found in prokaryotes; yet, it differs from this class of peptides in several important aspects. DAP is a tridecapeptide, in which the 10 C-terminal amino acids are cyclized. The N-terminal amino acid, tryptophan, is amidated with n-decanoic acid, which enhances binding to lipid bilayers (16). The amino acid composition is unusual in that two residues, 3-methyglutamate and the highly fluorescent species kynurenine, are nonproteinogenic. In contrast to linear, alpha-helical peptides, native DAP binds to lipid bilayers only in the presence of calcium, and its activity is absolutely calcium dependent (13,17).DAP is clinically important in the treatment of multidrug-resistant infections, including those caused by methicillin-resistant S. aureus (MRSA) strains. It is unclear if the binding of DAP to lipid bil...

Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains

“…Loss-of-function mutations in the PG synthase PgsA and gainof-function mutations in the lysyl-PG synthase MprF lead to a reduction in negatively charged PG in the membrane. PG is needed for daptomycin-membrane interaction and antibacterial activity (81,(84)(85)(86). Mutations in the cardiolipin synthase Cls2 are associated with daptomycin resistance in S. aureus, Enterococcus faecalis, and E. faecium (81).…”

The pH-dependence of lipid-mediated antimicrobial peptide resistance in a model staphylococcal plasma membrane: A two-for-one mechanism of epithelial defence circumvention

“…Various biophysical studies using synthetic lipid vesicles have attempted to demonstrate this charge dampening mechanism, with somewhat equivocal results. Experiments using both labile (Danner, et al 2008) synthetic native LPG and its chemically stable analogue LPE have shown that although they attenuate AMP-induced membrane lysis, significant amounts of peptide remained bound to the lipid bilayers (Kilelee, et al 2010, Khatib, et al 2016. None of these studies, however, had considered the role of mild acidity as an activator of LPG-mediated membrane protection, through inducing increased LPG biosynthesis (Gould and Lennarz 1970), eliciting demonstrable attenuation of peptide binding (Rehal, et al 2017) and thus significant resistance to AMPs in S. aureus (Walkenhorst, et al 2013).…”

Environment Shapes the Accessible Daptomycin Resistance Mechanisms inEnterococcus faecium

“…Similarly, MprF catalyzes the transfer of a lysyl group to phosphatidylglycerol causing a net increase in cell surface charge and reduced DAP binding (18). Although, it has also been postulated that this reaction affects DAP susceptibility by decreasing the availability of phosphatidylglycerol for DAP binding (24). In contrast, Efc employs a different strategy that involves redistribution of anionic phospholipid microdomains away from the septum, to divert DAP from critical septal targets; this mechanism is mediated by the LiaFSR cell-envelope-stress-response system in association with cardiolipin synthase ( cls ) (15, 25, 26).…”