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A Tandem Liquid Chromatography–Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus
Published on: March 28, 2017
Antibiotic Effects on Methicillin-Resistant Staphylococcus aureus Cytoplasmic Peptidoglycan Intermediate Levels and
Harika Vemula1, Navid J Ayon1, Alloch Burton1
1Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri, USA.
Abstract:
Cytoplasmic peptidoglycan (PG) precursor levels were determined in methicillin-resistant Staphylococcus aureus (MRSA) after exposure to several cell wall-targeting antibiotics. Three experiments were performed: (i) exposure to 4× MIC levels (acute); (ii) exposure to sub-MIC levels (subacute); (iii) a time course experiment of the effect of vancomycin. In acute exposure experiments, fosfomycin increased UDP-GlcNAc, as expected, and resulted in substantially lower levels of total UDP-linked metabolite accumulation relative to other pathway inhibitors, indicating reduced entry into this pathway. Upstream inhibitors (fosfomycin, d-cycloserine, or d-boroalanine) reduced UDP-MurNAc-pentapeptide levels by more than fourfold. Alanine branch inhibitors (d-cycloserine and d-boroalanine) reduced d-Ala-d-Ala levels only modestly (up to 4-fold) but increased UDP-MurNAc-tripeptide levels up to 3,000-fold. Downstream pathway inhibitors (vancomycin, bacitracin, moenomycin, and oxacillin) increased UDP-MurNAc-pentapeptide levels up to 350-fold and UDP-MurNAc-l-Ala levels up to 80-fold, suggesting reduced MurD activity by downstream inhibitor action. Sub-MIC exposures demonstrated effects even at 1/8× MIC which strongly paralleled acute exposure changes. Time course data demonstrated that UDP-linked intermediate levels respond rapidly to vancomycin exposure, with several intermediates increasing three- to sixfold within minutes. UDP-linked intermediate level changes were also multiphasic, with some increasing, some decreasing, and some increasing and then decreasing. The total (summed) UDP-linked intermediate pool increased by 1,475 μM/min during the first 10 min after vancomycin exposure, providing a revised estimate of flux in this pathway during logarithmic growth. These observations outline the complexity of PG precursor response to antibiotic exposure in MRSA and indicate likely sites of regulation (entry and MurD).
Insights
Antibiotic exposure alters peptidoglycan precursor levels in MRSA, revealing key regulation points. Understanding these changes helps in developing new strategies against resistant bacteria.
Area of Science:
- Microbiology
- Biochemistry
- Pharmacology
Background:
- Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant public health threat.
- Understanding the mechanisms of antibiotic action is crucial for developing new therapeutic strategies.
- Peptidoglycan (PG) synthesis is a key target for many cell wall-targeting antibiotics.
Purpose of the Study:
- To investigate the impact of various cell wall-targeting antibiotics on cytoplasmic peptidoglycan precursor levels in MRSA.
- To identify potential regulatory sites in the PG synthesis pathway in response to antibiotic stress.
- To characterize the dynamic changes in PG precursor pools under acute, sub-acute, and time-course antibiotic exposure.
Main Methods:
- Exposure of MRSA to different concentrations (4x MIC and sub-MIC) of antibiotics including fosfomycin, d-cycloserine, d-boroalanine, vancomycin, bacitracin, moenomycin, and oxacillin.
- Quantitative analysis of cytoplasmic UDP-linked peptidoglycan precursors using liquid chromatography-tandem mass spectrometry.
- Time-course experiments to monitor the rapid changes in precursor levels following vancomycin exposure.
Main Results:
- Upstream inhibitors (fosfomycin, d-cycloserine, d-boroalanine) significantly reduced UDP-MurNAc-pentapeptide levels and altered UDP-linked metabolite accumulation.
- Alanine branch inhibitors (d-cycloserine, d-boroalanine) increased UDP-MurNAc-tripeptide levels, indicating potential feedback regulation.
- Downstream inhibitors (vancomycin, bacitracin, moenomycin, oxacillin) elevated UDP-MurNAc-pentapeptide and UDP-MurNAc-l-Ala levels, suggesting inhibition of MurD activity.
- Sub-MIC exposures mirrored acute exposure effects, highlighting sensitivity even at low antibiotic concentrations.
- Vancomycin exposure induced rapid, multiphasic changes in UDP-linked intermediate levels, with a significant increase in total precursor pool within minutes.
Conclusions:
- Antibiotic exposure triggers complex and dynamic responses in MRSA's peptidoglycan precursor pools.
- The study identifies entry into the pathway and MurD activity as likely critical sites of regulation.
- These findings provide valuable insights into MRSA's adaptive strategies and potential targets for novel antibiotic development.
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