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Journal of Lipid Research, Vol. 48, 2419-2427, November 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

A rapid, small-scale procedure for the structural characterization of lipid A applied to Citrobacter and Bordetella strains: discovery of a new structural element

Alina Tirsoaga^1,*, Asmaa El Hamidi^*, Malcolm B. Perry, Martine Caroff^* and Alexey Novikov^2,*

^* Equipe Endotoxines, Unité Mixte de Recherche 8619 du Centre National de la Recherche Scientifique, Institut de biochimie et biophysique moléculaire et cellulaire, Université de Paris-Sud, Orsay, France
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada

Published, JLR Papers in Press, August 16, 2007.

¹ Present address of A. Tirsoaga: Department of Physical Chemistry, University of Bucharest, Bucharest, Romania.

² To whom correspondence should be addressed. e-mail: alexey.novikov{at}u-psud.fr

Endotoxins [lipopolysaccharides (LPSs)] are part of the outer cell membrane of Gram-negative bacteria. Their biological activities are associated mainly with the lipid component (lipid A) and even more specifically with discrete aspects of their fine structure. The need for a rapid and small-scale analysis of lipid A motivated us to develop a procedure that combines direct isolation of lipids A from bacterial cells with sequential release of their ester-linked fatty acids by a mild alkali treatment followed by MALDI-MS analysis. This method avoids the multiple-step LPS extraction procedure and lipid A isolation. The whole process can be performed in a working day and applied to lyophilized bacterial samples as small as 1 mg. We illustrate the method by applying it to the analysis of lipids A of three species of Citrobacter that were found to be identical. On the other hand, when applied to two batches of Bordetella bronchiseptica strain 4650, it highlighted the presence, in one of them, of hitherto unreported hexosamine residues substituting the lipid A phosphate groups, possibly a new camouflage opportunity to escape a host defense system.

Supplementary key words de-O-acylation • Gram-negative bacteria • hexosamine • endotoxin • lipopolysaccharide • mass spectrometry

Abbreviations: C₁₆, hexadecanoic acid; C₁₂, dodecanoic acid; C₁₄, tetradecanoic acid; C₁₄-OH, hydroxytetradecanoic acid; GlcN, glucosamine; HF, hydrofluoric acid; LPS, lipopolysaccharide

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