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GNPS - Defective pgsA contributes to increased membrane fluidity and cell wall thickening in S. aureus with high-level daptomycin resistance

ABSTRACT: Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations 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 isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported 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. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. RT-qPCR studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels.

INSTRUMENT(S): Synapt XS

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

SUBMITTER: Kelly M. Hines

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

REPOSITORIES: GNPS

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Project description:The discovery of antibiotics has led to the effective treatment of bacterial infections that were otherwise fatal and has had a transformative effect on modern medicine. Teixobactin is an unusual depsipeptide natural product that was recently discovered from a previously unculturable soil bacterium which is active against several Gram-positive pathogens, including methicillin- resistant Staphylococcus aureus and vancomycin-resistant Enterococci. One of the key attractive features of teixobactin as an antibiotic lead is that resistance could not be generated in a laboratory setting against S. aureus. This is proposed to be a result of the mechanism of action that involves binding to essential bacterial cell wall synthesis building blocks lipid II and lipid III.In the present study, metabolomics was used to investigate the potential metabolic pathways and mode of action involved in mechanisms of antibacterial activity and bacterial killing of the synthetic teixobactin analog Leu10-teixobactin against an MRSA strain, S. aureus ATCC700699. The metabolomes of S. aureus ATCC700699 cells were compared at 1, 3 and 6 h following treatment with Leu10-teixobactin (0.5 µg/ml, i.e. 0.5x MIC) and the untreated controls. Leu10-teixobactin significantly perturbed bacterial membrane lipids (glycerophospholipids and fatty acids), peptidoglycan (lipid I and II) metabolism and cell wall teichoic acid (lipid III) biosynthesis as early as after 1 h of treatment reflecting an initial activity on the cell envelope. Concordant with its time-dependent antibacterial killing action, Leu10-teixobactin caused more perturbations in the levels of key intermediates in pathways of amino-sugar and nucleotide-sugar metabolism and their downstream peptidoglycan and teichoic acid biosynthesis at 3 and 6 h. Significant perturbations in arginine metabolism and interrelated tricarboxylic acid cycle, histidine metabolism, pantothenate and Coenzyme A biosynthesis were also observed at 3 and 6 h.To conclude, this is the first study to provide novel metabolomics mechanistic information which lends to support the development of teixobactin as an antibacterial drug for the treatment of multi-drug resistant Gram-positive infections.

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GNPS - Defective pgsA contributes to increased membrane fluidity and cell wall thickening in S. aureus with high-level daptomycin resistance

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