Daptomycin is an important, last-resort antimicrobial therapeutic for the treatment of infections caused by Staphylococcus aureus that has acquired β-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 β -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 .The cationic lipopeptide antimicrobial daptomycin has become an essential tool for combating infections with Staphylococcus aureus that display reduced susceptibility to beta lactams or vancomycin. Since the mechanism of daptomycin's activity is based on interaction with the negatively charged membrane of S. aureus, the most commonly detected routes to daptomycin-resistance occur through SNPs in the lipid biosynthetic pathway surround phosphatidylglycerols and regulatory system that control cell envelope homeostasis. We demonstrate that a strain of MRSA N315 with high-level daptomycin resistance due to mutations in pgsA, yycG , and mprF has aberrantly high membrane fluidity and thickened cell. These phenotypes can be reserved upon supplementation of the culture broth with exogenous SCFAs through their incorporation through the FakA pathway. Our results give premise to the concept that targeted remodeling of the staphylococcal membrane may be an advantageous strategy to restore daptomycin susceptibility.

Increased membrane fluidity and cell wall thickening contribute to high-level daptomycin resistance in S. aureus with defective pgsA and yycG