1. Targeted lipopolysaccharide biosynthetic intermediate analysis with normal-phase liquid chromatography mass spectrometry
- Author
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Brian Y. Feng, Tsuyoshi Uehara, Ramadevi Prathapam, Mina Mostafavi, Pramila Tamrakar, Lisha Wang, Christopher M. Rath, William S. Sawyer, and Louis E. Metzger
- Subjects
Lipopolysaccharides ,Lipopolysaccharide ,Artificial Gene Amplification and Extension ,medicine.disease_cause ,Pathology and Laboratory Medicine ,Biochemistry ,Polymerase Chain Reaction ,chemistry.chemical_compound ,Liquid chromatography–mass spectrometry ,Antibiotics ,Medicine and Health Sciences ,chemistry.chemical_classification ,0303 health sciences ,Multidisciplinary ,biology ,Chemistry ,Antimicrobials ,Drugs ,Pseudomonas Aeruginosa ,Lipids ,Bacterial Pathogens ,Medical Microbiology ,Periplasm ,Medicine ,lipids (amino acids, peptides, and proteins) ,Metabolic Pathways ,Pathogens ,Bacterial outer membrane ,Research Article ,Cell Physiology ,Gram-negative bacteria ,Science ,Research and Analysis Methods ,Biosynthesis ,Microbiology ,03 medical and health sciences ,Microbial Control ,Pseudomonas ,medicine ,Escherichia coli ,Molecular Biology Techniques ,Gram Negative Bacteria ,Molecular Biology ,Microbial Pathogens ,030304 developmental biology ,Pharmacology ,Bacteria ,030306 microbiology ,Organisms ,Biology and Life Sciences ,Bacteriology ,Periplasmic space ,Cell Biology ,biology.organism_classification ,Cell Metabolism ,Metabolic pathway ,Enzyme ,Metabolism ,Acyltransferases - Abstract
Lipopolysacharride (LPS) forms the outer leaflet of the outer membrane in Gram-negative bacteria and contributes to the permeability barrier and immune response. In this study, we established a method for monitoring the LPS biosynthetic intermediates of the Raetz pathway (lpxA-lpxK) in Escherichia coli. Metabolites from compound-treated cells and genetically-perturbed cells were extracted from whole cells and concentrated by mixed-mode weak anion exchange (WAX) solid-phase extraction (SPE) prior to analysis by normal phase (NP)LC-MS/MS. Data was normalized to cell density and an internal standard prior to comparison against untreated cells in order to determine fold accumulation and depletion for affected metabolites. Using this LC-MS/MS method, we were able to reliably monitor changes in levels of the LPS intermediates in response to compound-treatment and genetic modification. In addition, we found that deletion of periplasmic CDP-diacylglycerol pyrophosphatase dramatically increased levels of the UDP-containing LPS intermediates, suggesting the enzymatic breakdown during sample preparation. This assay allows for probing a key essential pathway in Gram-negative bacteria in an effort to discover antibacterial agents that inhibit enzymes in the LPS biosynthetic pathway.
- Published
- 2019