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18th Annual Conference of the Metabolomics Society

Combining Cellular Bioenergetics with Metabolomics

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Agilent Lunch Seminar - Wed 22nd June at 12:20pm

Title: Deciphering the Mechanisms of Immunometabolism in Eukaryotes and Drug Resistance in Bacteria using Extracellular Flux Analysis and 13C stable-Isotope Tracing

Speaker: Dr Gerald Larrouy-Maumus, Senior Lecturer, Imperial College London


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We look forward to welcoming you to Booth #4 at this year's Metabolomics Society in-person meeting. Our technical experts will be available at the booth to discuss solutions with you, and to talk you through the Agilent portfolio of instruments and consumables. Visit us throughout the conference to learn more on:

  • Bravo Metabolomics Sample Prep Automation for increased throughput, consistency, and reproducibility
  • Easy-to-Implement targeted Metabolomics & Lipidomics strategies for high-sensitivity
  • Powerful High-Resolution and Flux approaches for discovery analysis
  • Comprehensive Cellular Analysis solutions to decipher biological insights at the functional level
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Bioenergetics and metabolism are intimately connected. However, how those two can be executed and integrated can represent a challenge. In this presentation, I will discuss the experimental design, analytical methods and interpretation of the data generated for two examples taken from our laboratory.  

The first one will uncover the changes in metabolism upon LPS stimulation of macrophages. Indeed, eukaryotic cells are known to generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Agilent Seahorse technology is an invaluable tool to gain access in real time to the bioenergetics activity of the cell by measuring cellular oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as measures of mitochondrial respiration and glycolysis, respectively. Even though Seahorse provides phenotypic information about the bioenergetic state of the cell, an in-depth understanding of the changes in the metabolic pathways requires measuring the abundances and rates of metabolite interconversion of the molecules involved in the key metabolic pathways (e.g. glycolysis, tricarboxylic acid cycle (TCA) cycle, pentose phosphates pathway). I will present an example of using both Seahorse XF technology and 13C stable-isotope-tracing analysis with Agilent MassHunter VistaFlux software to study the response to lipopolysaccharide (LPS) treatment of RAW 264.7 macrophages. 

The second example will focus on how Seahorse bioenergetics combined to stable-isotope tracing helped to decipher the mechanisms by which mycobacteria become tolerant to antibiotics. Antimicrobial tolerance is the gateway to the development of antimicrobial resistance (AMR) and is therefore a major issue that needs to be addressed. The second messenger 3’,5’-cyclic adenosine monophosphate (cAMP), which is conserved across all taxa, is involved in propagating signals from environmental stimuli and converting these signals into a response. However, cAMP signalling in mycobacteria is particularly complex, making the investigation of cAMP signalling and its involvement in antimicrobial tolerance difficult. To address this pressing need, we identified a new cyclic nucleotides degrading phosphodiesterase enzyme (Rv1339) and used it as a tool to significantly decrease cAMP levels in mycobacteria. This analysis revealed that in Mycobacterium smegmatis mc2155, the expression of Rv1339 reduced cAMP levels, altered genes expression leading to impaired bioenergetics, increased peptidoglycan turnover and led to decreased tolerance to antimicrobials that target cell wall synthesis. By combining Seahorse bioenergetics and LC/MS based 13C stable isotope tracing, this work represents an important milestone by showing that targeting nucleotide signalling is a promising new avenue for antimicrobial development and expands our understanding of nucleotide signalling in mycobacteria.