Abstract
Monophosphoryl lipid A was prepared from the lipopolysaccharide of Chlamydia trachomatis, converted to the methyl ester, and fractionated by reverse-phase high-performance liquid chromatography. The peak fractions were collected and analyzed by mass spectrometry. Matrix-assisted laser desorption/ionization and liquid secondary ion mass spectrometry of the first of two major high-performance liquid chromatographic fractions showed multiple quasi-molecular ions of MNa+ at m/z 1780, 1794, 1808, 1822, and 1836. The positive-ion liquid secondary ion mass spectrometry spectrum also showed a minor series of peaks at m/z 1916, 1930, 1944, 1958, and 1971, consistent with the formation of matrix adducts with 3-nitrobenzyl alcohol. Oxonium ions representing the distal subunit were observed at m/z 1057, 1071, 1085, 1099, and 1113. The second fraction was similarly analyzed and found to contain structural homologs of the first fraction. Based on this study, the major lipid A component of chlamydial lipopolysaccharide is a glucosamine disaccharide that contains five fatty acids and a phosphate in the distal segment. Three fatty acyl groups are in the distal segment, and two are in the reducing end segment. The acyloxyacyl group is located in the distal segment in amide linkage. Two structural series, differing by 14 atomic mass units in the reducing subunit, were observed. Chlamydial lipid A is complex and consists of at least 20 homologous structural components. The relatively low potency of Chlamydia trachomatis lipopolysaccharide in activating lipopolysaccharide-responsive cells might be related to the unusual fatty acid composition of the lipid A moiety.
Highlights
Bacteria of the genus Chlamydia are pathogenic, obligatory phagosomal intracellular parasites that cause acute and chronic diseases in animals and humans [1, 2]
When C. trachomatis is grown in tissue culture, chlamydial LPS appears to lack the characteristic O antigen that is observed in LPS from other Gram-negative bacteria
Preparation and Characterization of MPLA Derived from LPS of C. trachomatis—The LPS of C. trachomatis was acidhydrolyzed to yield crude MPLA, which was converted to the free acid and methylated with diazomethane
Summary
Materials—HPLC-grade chloroform, methanol, acetonitrile, and isopropyl alcohol were purchased from Burdick & Jackson (Muskegon, MI). The MPLA (6 mg) was converted into the free acid form by passing through Chelex 100 (Naϩ) and Dowex 50 (Hϩ) columns, as described previously [12], and methylated with diazomethane. The fatty acids were extracted and derivatized to the methyl or converted to the p-bromophenacyl esters as described previously [16]. The p-bromophenacyl esters of the fatty acids were analyzed by reverse-phase HPLC as described previously [16]. MS of Purified Dimethyl-MPLA—Liquid secondary ion mass spectrometry (LSIMS) spectra of HPLC-purified dimethylated MPLA were acquired on a Concept IH two-sector mass spectrometer All matrix-assisted laser desorption/ionization/ion trap (MALDI/IT) mass spectra were acquired on an extensively modified Finnigan MAT quadrupole ion trap mass spectrometer described elsewhere [20]. Spectra acquired from the ion trap were processed using a TOFware® data system (Ilys Software, Pittsburgh, PA)
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