Up to 80% of the fatty acids in Staphylococcus aureus membrane lipids are branched, rather than straight-chain, fatty acids. The branched fatty acids (BCFAs) may have either an even or odd number of carbons, and the branch position may be at the penultimate carbon (iso) or the antepenultimate (anteiso) carbon of the tail. This results in two sets of isomeric fatty acid species with the same number of carbons that cannot be resolved by mass spectrometry. The isomer/isobar challenge is further complicated when the mixture of BCFAs and straight-chain fatty acids (SCFAs) are esterified into diacylated lipids such as the phosphatidylglycerol (PG) species of the S. aureus membrane. No conventional chromatographic method has been able to resolve diacylated lipids containing mixtures of SCFAs, anteiso-odd, iso-odd, and iso-even BCFAs. A major hurdle to method development in this area is the lack of relevant analytical standards for lipids containing BCFA isomers. The diversity of the S. aureus lipidome and its naturally high levels of BCFAs present an opportunity to explore the potential of resolving diacylated lipids containing BCFAs and SFCAs. Using our knowledge of lipid and fatty acid biosynthesis in S. aureus, we have used a stable-isotope-labeling strategy to develop and validate a 30 min C18 reversed-phase liquid chromatography method combined with traveling-wave ion mobility–mass spectrometry to provide resolution of diacylated lipids based on the number of BCFAs that they contain.
Daptomycin is an important, last-resort antimicrobial therapeutic for the treatment of infections caused by Staphylococcus aureus that has acquired beta-lactam nonsusceptibility or reduced vancomycin susceptibility. The mechanism of action of daptomycin involves disruption of the cell membrane rather than cell wall synthesis, as with beta-lactams and vancomycin. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to single-nucleotide polymorphisms directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant strain selected from methicillin-resistant S. aureus (MRSA) N315 with mutations in the most commonly reported SNPs associated with daptomycin-resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin-resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness relative to its parental strain. Despite the enrichment of BCFAs, we found that the daptomycin-resistant strain successfully utilized isotope-labeled straight-chain fatty acids (SCFAs) in the synthesis of membrane lipids and that supplementation of the culture broth with SCFAs restored membrane fluidity in the daptomycin-resistant strain to the state of its parental strain. These results demonstrate that exogenous fatty acids can mitigate, in part, the phenotypes associated with daptomycin resistance when it is driven by mutations in yycG and pgsA.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.