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Updated: May 25, 2026
Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria
Published on: September 16, 2013
LC-MS/MS characterization of phospholipid content in daptomycin-susceptible and -resistant isolates of Staphylococcus
Aileen Rubio1, Jeff Moore, Mustafa Varoglu
1Cubist Pharmaceuticals, 65 Hayden Avenue, Lexington, MA 02421, USA. aileen.rubio@cubist.com
Abstract:
Daptomycin (DAP) is a cyclic lipopeptide antibiotic used for the treatment of certain Staphylococcus aureus infections. Although rare, strains have been isolated that are DAP resistant. These strains usually have mutations in mprF, a gene encoding a membrane protein with both lysylphosphatidylglycerol (LPG) synthase and flippase activities. Because ΔmprF strains have increased DAP susceptibility, the mechanism of resistance is not likely due to a loss of mprF function. In this study, we developed an LC-MS assay to examine the effect of different mprF mutations on the ratio of phosphatidylglycerol (PG) to LPG in the membrane. Our assay demonstrated that some, but not all, mutations in the flippase and synthase domains result in small but reproducible increases in the proportion of LPG relative to PG. Techniques described herein represent a higher throughput and more sensitive method for measuring relative phospholipids levels. These results offer guidance in the understanding of how mprF confers DAP resistance; namely, mprF-mediated resistance may be through more than one mechanism, including increased overall LPG synthesis and increased LPG present on the outer leaflet of the cytoplasmic membrane.
Insights
Daptomycin resistance in Staphylococcus aureus involves mutations in the mprF gene. This study shows mprF mutations can increase lysylphosphatidylglycerol (LPG) in bacterial membranes, contributing to daptomycin resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Daptomycin (DAP) is a critical antibiotic for treating Staphylococcus aureus infections.
- Daptomycin resistance is an emerging clinical challenge, often linked to mutations in the mprF gene.
- The mprF gene encodes a protein involved in lysylphosphatidylglycerol (LPG) synthesis and membrane lipid transport.
Purpose of the Study:
- To investigate the impact of mprF mutations on the ratio of phosphatidylglycerol (PG) to LPG in bacterial membranes.
- To develop a sensitive method for quantifying relative phospholipid levels.
Main Methods:
- Development of a liquid chromatography-mass spectrometry (LC-MS) assay.
- Analysis of phospholipid composition in bacterial strains with various mprF mutations.
- Quantification of phosphatidylglycerol (PG) and lysylphosphatidylglycerol (LPG) ratios.
Main Results:
- Some mprF mutations led to a measurable increase in the proportion of LPG relative to PG.
- The developed LC-MS assay offers higher throughput and sensitivity for phospholipid analysis.
- Not all mprF mutations resulted in altered LPG/PG ratios, suggesting complex resistance mechanisms.
Conclusions:
- mprF-mediated daptomycin resistance may involve multiple mechanisms.
- Increased synthesis of LPG and its presence on the outer membrane leaflet are potential contributors to resistance.
- Understanding these mechanisms can guide strategies to combat antibiotic resistance.
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