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Structure of a novel lipid A obtained from the lipopolysaccharide of Caulobacter crescentus

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada

Igor A. Kaltashov

The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Robert J. Cotter

The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Evgeny Vinogradov

Institute for Biological Sciences, National Research Council, Ottawa, Canada

Malcolm B. Perry

Institute for Biological Sciences, National Research Council, Ottawa, Canada

Hibba Haider

Department of Basic Medical Science, School of Medicine, and Shock/Trauma Research Center, University of Missouri, Kansas City, Missouri, USA

Nilofer Qureshi

Department of Basic Medical Science, School of Medicine, and Shock/Trauma Research Center, University of Missouri, Kansas City, Missouri, USA, qureshin{at}umkc.edu

Caulobacter crescentus CB15 is a dimorphic bacterium that is best known as a prokaryotic model for cell development. However, it is also being exploited in biotechnology, where the crystalline surface (S-layer) protein secretion system has been adapted for heterologous protein display or secretion. Because the S-layer attaches to the cell surface via lipopolysaccharide (LPS) and since the LPS represents a potential endotoxin contaminant of recombinant proteins, the lipid A component was examined in detail. LPS was acid hydrolyzed to obtain crude lipid A, which was methylated and purified by HPLC. HPLC peak fractions were analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy. The structure of the major lipid A of C. crescentus comprised the tetrasaccharide backbone -D-GalpA-(14)-β-D-DAG-(16)--D-DAG-(11)--D-GalpA substituted with six fatty acids, and a molecular mass of 1875 (GalpA, galactopyranuronic acid; DAG, 2,3-diamino-2,3-dideoxyglucopyranose). No phosphate residues were detected. The major lipid A component had 12:0[3-O[⁵-12:1(3-OH)]] and 12:0[3-O(⁵-12:1)] fatty acyl chains at either the 3'- or the 2' positions of the distal subunit DAG B, and 12:0(3OH) and 12:0[3,6-(OH) ₂] fatty acyl chains at 3- and 2- positions of the reducing end subunit DAG A, respectively. In addition, several other variations in the structure were observed. The LPS was evaluated for TNF- inducing activity and consistent with its unusual lipid A structure (relative to that of enteric bacteria), the activity was reduced by greater than 100-fold as compared to Escherichia coli ReLPS. This and other evidence suggests the potential application of this lipid A as a vaccine adjuvant or the suitability of Caulobacter displaying antigens for formulation of whole cell vaccines.

Key Words: Lipid A • lipopolysaccharide • Caulobacter crescentus

Innate Immunity, Vol. 14, No. 1, 25-36 (2008)
DOI: 10.1177/1753425907087588


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