Targeted lipopolysaccharide biosynthetic intermediate analysis with normal-phase liquid chromatography mass spectrometry

PLoS One. 2019 Feb 8;14(2):e0211803. doi: 10.1371/journal.pone.0211803. eCollection 2019.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / genetics
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Lipopolysaccharides / biosynthesis*
  • Lipopolysaccharides / genetics
  • Periplasm / genetics
  • Periplasm / metabolism*

Substances

  • Lipopolysaccharides
  • Acyltransferases
  • acyl-(acyl-carrier-protein)-UDP-N-acetylglucosamine acyltransferase

Grants and funding

All funding for this project was supplied by the Novartis Institutes for Biomedical Research. The funder provided support in the form of salaries for all authors, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Current employers listed, such as Genentech, Tierra Biosciences, and AbSci were not involved with the research in any way, and are listed for purposes of identifying and contacting the authors only.