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Micromethods for Isolation and Structural Characterization of Lipid A, and Polysaccharide Regions of Bacterial Lipopolysaccharides

  • Alexey Novikov
  • Aude Breton
  • Martine Caroff
Part of the Methods in Molecular Biology book series (MIMB, volume 1600)

Abstract

Lipopolysaccharides (LPS) are major components of the external membrane of most Gram-negative bacteria, providing them with an effective permeability barrier. They are essentially composed of a hydrophilic polysaccharide region (PS) linked to a hydrophobic one, termed lipid A. The LPS polysaccharide moiety is divided into the core oligosaccharide (OS) and O-chain repetitive elements. Depending on their individual variable fine structures, LPS may be potent immunomodulators. The lipid A structure is a key determinant for LPS activity. However, the presence of the core region, or at least of the highly charged 3-deoxy-d-manno-oct-2-ulosonic acid molecules, is also important for preserving the native lipid A conformation within individual LPS molecules. We describe herein four rapid and practical micromethods for LPS, lipid A, and core OS structural analyses. The first method allows the direct isolation of lipid A from whole bacteria cell mass; the second describes conditions for the sequential release of fatty acids enabling the characterization of their substitution position in the lipid A backbone, to be determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The third one is a microscale procedure for the mass spectra screening of LPS, lipid A, and PS using triethylamine and citric acid. The fourth method is a chromatography procedure for Rough-type LPS on thin-layer-chromatography. These methods were developed to be coupled to mass-spectrometry (e.g., MALDI-MS) but can also be used with other analytical techniques (e.g., chromatography). Examples are given with reference to two major human pathogens: Bordetella pertussis and Pseudomonas aeruginosa; to one porcine pathogen: Actinobacillus pleuropneumoniae; and to commercial samples of Salmonella Minnesota Re595 LPS.

Key words

Gas chromatography 3-Deoxy-d-manno-oct-2-ulosonic acid Lipooligosaccharide Lipopolysaccharide Matrix-assisted laser desorption/ionization mass spectrometry Polyacrylamide gel electrophoresis Sodium dodecyl sulfate Triethylamine Thin-layer chromatography 

Notes

Acknowledgments

Aude Breton is a recipient of a CIFRE grant in collaboration with the LPS-BioSciences company. We thank Pr. Gottschalk for his kind exchanges and expertise with App strains and isolates.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.LPS-BioSciences, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-SudUniversité Paris-SaclayOrsayFrance

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