Daptomycin-Resistant Enterococcus faecalis Diverts the Antibiotic Molecule from the Division Septum and Remodels Cell Membrane Phospholipids

Abstract

Treatment of multidrug-resistant enterococci has become a challenging clinical problem in hospitals around the world due to the lack of reliable therapeutic options. Daptomycin (DAP), a cell membrane-targeting cationic antimicrobial lipopeptide, is the only antibiotic with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). However, the clinical use of DAP against VRE is threatened by emergence of resistance during therapy, but the mechanisms leading to DAP resistance are not fully understood. The mechanism of action of DAP involves interactions with the cell membrane in a calcium-dependent manner, mainly at the level of the bacterial septum. Previously, we demonstrated that development of DAP resistance in vancomycin-resistant Enterococcus faecalis is associated with mutations in genes encoding proteins with two main functions, (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase). In this work, we show that these VRE can resist DAP-elicited cell membrane damage by diverting the antibiotic away from its principal target (division septum) to other distinct cell membrane regions. DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis). Full expression of DAP resistance requires additional mutations in enzymes (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase) that alter cell membrane phospholipid content. Our findings describe a novel mechanism of bacterial resistance to cationic antimicrobial peptides.IMPORTANCE The emergence of antibiotic resistance in bacterial pathogens is a threat to public health. Understanding the mechanisms of resistance is of crucial importance to develop new strategies to combat multidrug-resistant microorganisms. Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. Daptomycin (DAP) is a calcium-decorated antimicrobial lipopeptide whose target is the bacterial cell membrane. A current paradigm suggests that Gram-positive bacteria become resistant to cationic antimicrobial peptides via an electrostatic repulsion of the antibiotic molecule from a more positively charged cell surface. In this work, we provide evidence that VRE use a novel strategy to avoid DAP-elicited killing. Instead of “repelling” the antibiotic from the cell surface, VRE diverts the antibiotic molecule from the septum and “traps” it in distinct membrane regions. We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.

The action mechanism of daptomycin

“…66 However, in the liposome model at least, CL inhibits membrane permeabilization, 66 which suggests that it might mediate resistance to daptomycin by competing for it with PG. Such an effect has indeed been claimed to occur in vivo, 67 but the CL-enriched membrane domains purported in that study were identified by staining with nonyl-acridine orange, which has since been shown to bind to phosphatidylglycerol also. 68 Thus, no conclusive evidence is available to support a preferential interaction between daptomycin and CL in vivo (see also section 12).…”

PhoPQ regulates acidic glycerophospholipid content of the Salmonella Typhimurium outer membrane

“…Therefore, CL molecules may direct CAMP, or cell division components, to specific microdomains to increase bacterial resistance, or coordinate membrane fission with OM lipid A and PG remodeling. In support of a role for CL in CAMP resistance, vancomycin-resistant Gram-positive Enterococcus faecalis can clinically acquire daptomycin resistance by sequestering CAMP to distinct negatively charged CL microdomains within the plasma membrane (33). In addition, electron micrographs of antimicrobial peptides interacting with bacterial surfaces have captured initial membrane-active events (34).…”

Penicillin Binding Protein 1 Is Important in the Compensatory Response of Staphylococcus aureus to Daptomycin-Induced Membrane Damage and Is a Potential Target for β-Lactam–Daptomycin Synergy

“…The activity of DAP is dependent not only on the quantity of binding but also on the quality of binding, as some ␤-lactams induce marked increases in DAP surface binding but do not increase DAP potency. This feature has been recently highlighted in the mechanism of DAP resistance in Enterococcus faecalis, whereby DAP is bound and diverted to membrane sites away from the septum and, therefore, bound drug is sequestered to a site where it is functionally inactive in killing the bacterium (35). NAF, which is considered to be the optimal agent against serious methicillin-susceptible S. aureus infections, appears to exhibit a "best of both worlds" phenotype of potentiating the activity of both DAP and LL37.…”