Comprehensive analysis of phospholipids and glycolipids in the opportunistic pathogen Enterococcus faecalis

Abstract

Enterococcus faecalis is a Gram-positive, opportunistic, pathogenic bacterium that causes a significant number of antibiotic-resistant infections in hospitalized patients. The development of antibiotic resistance in hospital-associated pathogens is a formidable public health threat. In E. faecalis and other Gram-positive pathogens, correlations exist between lipid composition and antibiotic resistance. Resistance to the last-resort antibiotic daptomycin is accompanied by a decrease in phosphatidylglycerol (PG) levels, whereas multiple peptide resistance factor (MprF) converts anionic PG into cationic lysyl-PG via a trans-esterification reaction, providing resistance to cationic antimicrobial peptides. Unlike previous studies that relied on thin layer chromatography and spectrophotometry, we have performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) directly on lipids extracted from E. faecalis, and quantified the phospholipids through multiple reaction monitoring (MRM). In the daptomycin-sensitive E. faecalis strain OG1RF, we have identified 17 PGs, 8 lysyl-PGs (LPGs), 23 cardiolipins (CL), 3 glycerophospho-diglucosyl-diacylglycerols (GPDGDAG), 5 diglucosyl-diacylglycerols (DGDAG), 3 diacylglycerols (DAGs), and 4 triacylglycerols (TAGs). We have quantified PG and shown that PG levels vary during growth of E. faecalis in vitro. We also show that two daptomycin-resistant (DapR) strains of E. faecalis have substantially lower levels of PG and LPG levels. Since LPG levels in these strains are lower, daptomycin resistance is likely due to the reduction in PG. This lipidome map is the first comprehensive analysis of membrane phospholipids and glycolipids in the important human pathogen E. faecalis, for which antimicrobial resistance and altered lipid homeostasis have been intimately linked.

Membrane adaptation limitations inEnterococcus faecalisunderlie sensitivity and the inability to develop significant resistance to conjugated oligoelectrolytes

“…For example, for in vitro evolved daptomycin resistant E. faecalis strains, a 256-512 fold increase in MIC compared to the sensitive strain was reported. 21,30 Previous in vitro evolution studies with daptomycin (DAP) and resistant variants of E. faecalis V583 showed a "step pattern" of increase in DAP resistance over a 15 d period. This pattern was characterized by some of the isolates exhibiting daptomycin resistance of 128 mg mL À1 until day 11, 256 mg mL À1 until day 13, and quickly reaching peak in daptomycin resistance of 512 mg mL À1 on the day 14.…”

Characterization of the Mechanisms of Daptomycin Resistance among Gram-Positive Bacterial Pathogens by Multidimensional Lipidomics

“…Untargeted liquid chromatography-mass spectrometry (LC-MS) has also been applied to the analysis of bacterial lipids with improved sensitivity and specificity ( 36 ), but it remains difficult to resolve overlapping and similar-mass lipid species. More recently, Rashid et al used a combination of targeted and untargeted LC-MS methods for comprehensive lipidomic studies of E. faecalis , but the workflow is time-consuming as separate sample preparations and LC-MS methods are needed for glycerolipids, phospholipids, and CLs ( 37 ). To address the limitations of the previous methods, we recently developed a 3D lipidomics approach based on hydrophilic interaction LC-ion mobility-MS (HILIC-IM-MS) that enables high-throughput and high-specificity lipid analysis ( 38 ).…”

Enzyme-mediated photoinactivation of Enterococcus faecalis using Rose Bengal-acetate

“…3). We have previously observed an identical accumulation of RB at the cellular poles of E. faecalis, and suggested that the fluorescent spots observed reflect an affinity of the PS for cardiolipin, a phospholipid known to cluster into membrane domains at the poles of bacteria [7,26]. Such affinity was previously described by Tran et al [27] who used 10-nonyl acridine orange to stain the cardiolipin-rich domains of the E. faecalis membrane, and reported accumulation of the dye at the same spots as RB [27].…”