Staphylococcus aureus varies its membrane fluidity in response to environmental stresses by changing the ratio of branched-chain fatty acids (BCFAs) to straight-chain fatty acids (SCFAs) in its membrane lipids. Altered membrane fluidity has been associated with an increased tolerance of membrane-targeting antibiotics, including daptomycin. The routine assessment of microbial membrane fluidity relies on the measurement of total BCFA-versus-SCFA determination by GC-MS. Although GC-MS is capable of resolving BCFA and SCFA isomers, the requirement of free fatty acids eliminates the possibility to evaluate the preferences of lipid subclasses for BCFAs vs. SCFAs. We recently demonstrated a RPLC method that can separate lipid isomers having branched-branched, branched-straight, or straight-straight fatty acyl tail combinations. Although lipid classes co-eluted with the RPLC method, IM filtering in Skyline ensured high-quality fragmentation spectra for assignment of lipid identifications. Comparative lipidomics between S. aureus strains with and without daptomycin resistance revealed changes in both the absolute magnitude of lipid abundances and the distribution of FA isomers. As previously reported, N315-Dap8 had decreased levels of PGs and increased lysylPGs compared to N315 as a result of the pgsA and mrpF mutations. However, the RPLC-IM-MS method revealed that the PGs present in N315-Dap8 contained only branched-branched and branched-straight FA combinations, whereas N315 contained PGs with all three combinations of FAs. This shift in the structures of membrane phospholipids in the daptomycin-resistant organism is consistent with a dramatic increase in membrane fluidity to compensate for the overall reduction in PGs.

Assessing membrane fluidity of antibiotic-resistant Staphylococcus aureus using a RPLC-IM-MS method for isomeric phospholipid separations

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