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

ABSTRACT: 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.

INSTRUMENT(S): Synapt XS

ORGANISM(S): Staphylococcus Aureus (ncbitaxon:1280)

SUBMITTER: Kelly M. Hines

PROVIDER: MSV000091690 | MassIVE | Wed Apr 12 06:05:00 BST 2023

REPOSITORIES: MassIVE

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Project description:The phenotype “intermediate vancomycin resistance” in Staphylococcus aureus (CLSI: MIC = 4-8 mg/L) has been assigned to changes that lead to alterations in cell wall synthesis and morphology. Most vancomycin intermediately resistant S. aureus (VISA) strains are characterised by an increased cell wall thickness as a consequence of an activated cell wall biosynthesis and decreased autolysis. The purpose of this study was to analyse the genetic basis of the vancomycin resistance mechanism of the clinical VISA isolate SA137/93A and its spontaneous mutant strain SA137/93G. The methicillin-resistant S. aureus (MRSA) SA137/93A was isolated from a tracheal secretion and displays heterogeneous intermediate vancomycin resistance (hVISA strain, MIC: 8 mg/L in BHI). Subculturing in presence of 6 mg/L vancomycin generated a mutant with homogeneous intermediate vancomycin resistance, that showed an MIC value of 16 mg/L in BHI and was designated SA137/93G. PFGE profiles and phage typing of the strains showed that they were members of the Iberian clone (ST247), which was prevalent in Germany in the early nineties under the designation “Northern German epidemic strain”. Both strains possess a thickened cell wall. However, the vancomycin resistance of strain SA137/93A is most probably enhanced by an increased amount of free D-Ala-D-Ala termini in the cell wall, which is due to decreased crosslinking, whereas the mutant strain SA137/93G shows normal crosslinking. Moreover, strain SA137/93A displays an increased expression of the essential two-component system yycFGHI as a consequence of an IS256 insertion in the promoter region, while strain SA137/93G is characterised by an insertion of IS256 into the gene tcaA. Although both insertions were shown to correlate with a decrease in susceptibility to vancomycin, the difference in the vancomycin resistance level of the strain pair could be mainly attributed to the disruption of tcaA in the mutant.This study was conducted to identify resistance mechanisms that both strains might have in common. To this end we compared the transcriptomes of both strains with that of the closely related vancomycin susceptible MRSA/VSSA strain SA1450/94 (MIC: 2 mg/L). We found that the genes of the capsule biosynthesis were the only genes with higher expression in both VISA strains. Keywords: strain comparison

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

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