title: Structure of a novel lipid A obtained from the lipopolysaccharide of Caulobacter crescentus.
abstract: 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 alpha-D-GalpA-(1-->4)-beta-D-DAG-(1-->6)-alpha-D-DAG-(1-->1)-alpha-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[Delta(5)-12:1(3-OH)]] and 12:0[3-O(Delta(5)-12:1)] fatty acyl chains at either the 3'- or the 2' positions of the distal subunit DAG B, and 12:0(3-OH) 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-alpha 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.
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: John
authors.initials: J
authors.lastname: Smit
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kaltashov
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kaltoshov
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Cotter
authors.forename: Evgeny
authors.initials: E
authors.lastname: Vinogradov
authors.forename: Malcolm B
authors.initials: MB
authors.lastname: Perry
authors.forename: Hibba
authors.initials: H
authors.lastname: Haider
authors.forename: Nilofer
authors.initials: N
authors.lastname: Qureshi
mesh_terms: D000276:Adjuvants, Immunologic; D000818:Animals; D016935:Caulobacter crescentus; D002851:Chromatography, High Pressure Liquid; D008050:Lipid A / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051379:Mice; D008956:Models, Chemical; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D008050:Lipid A; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha