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.

FIG 1

BODIPY-labeled daptomycin (BDP-DAP) staining of representative E. faecalis S613 (DAP-susceptible) (a to e) and R712 (DAP-resistant) (f to j) cells at increasing concentrations of the fluorescent antibiotic. The images within each panel show bacterial cells captured by fluorescence (top image) and phase-contrast microscopy (bottom image) (bars, 1 µm). (a and b) In E. faecalis S613, binding of the antibiotic in a “peppering” pattern is observed only at subinhibitory concentrations of BDP-DAP. (c to e) At higher BDP-DAP concentrations, saturation of the surface is observed with staining of the division septum (white arrows). (f to j) In E. faecalis R712, the “peppering” staining pattern and binding of the fluorescent antibiotic is observed at all concentrations without visualization of septa. Additional images selected from different microscopic fields are shown in Fig. S2 in the supplemental material.

FIG 2

BODIPY-labeled DAP (BDP-DAP) staining of representative cells from E. faecalis S613 derivatives at increasing concentrations of the fluorescent antibiotic. The images for each BDP-DAP concentration (from 2 to 32 μg/ml) and strain show bacterial cells captured by fluorescence (top image) and phase-contrast (bottom image) microscopy (bars, 1 µm). (a) E. faecalis S613∆_liaF177 is a derivative of strain S613 carrying a mutated liaF belonging to E. faecalis R712 (daptomycin resistant). (b) S613∆_{liaF177gdpD170} is a derivative of S613 carrying both liaF and gdpD alleles from strain R712. (c) S613∆_{liaF177gdpD170cls61} carries mutated liaF, gdpD, and cls alleles from R712. Introduction of the three mutated alleles prevents BDP-DAP interaction with the septum up to concentrations of 16 µg/ml of BDP-DAP. Additional images selected from different microscopic fields are shown in Fig. S3 in the supplemental material. Arrows indicate visualization of septum in a linear pattern.

FIG 3

Staining of representative cells from E. faecalis S613 (DAP susceptible), R712 (DAP resistant), and S613 derivatives with 10-N-nonyl-acridine orange (NAO) at 500 nM revealing cardiolipin-rich domains in the cell membrane. The top images in panels show bacterial cells captured with a fluorescent microscope (bars, 1 µm). The bottom images in panels are phase-contrast images of the same bacterial cells. (a and d) In E. faecalis S613 and S613∆_gdpD170, CL-enriched domains are visualized at the poles and division septa. (b) CL-enriched domain redistribution is observed in DAP-resistant E. faecalis R712. (c) Introduction of a mutated liaF allele from strain R712 into strain S613 was sufficient to alter the distribution of CL domains. (e and f) Introduction of mutated gdpD and cls alleles to S613∆_liaF177 did not produce additional alterations in the distribution of CL-rich domains. Additional images selected from different microscopic fields are shown in Fig. S5 in the supplemental material.