Sialic acid and binding by the human pathogen Helicobacter pylori. Approaches to the structural analysis of complex glycosphingolipid mixtures and epitopes

Abstract

The human pathogen Helicobacter pylori colonizes the stomach of more than half of the global population and is implicated in gastric diseases such as chronic gastritis, gastric and duodenal ulcers and gastric carcinoma. The bacterium has to adhere to success and the aim of this study was to structurally characterize two different sialic acid dependent recognitions. The expression of binding specificities is dependent on growth conditions. Bacterial growth on solid media (agar plates) mediates expression of both specificities, while growth in liquid media (broth) mediates selective expression of recognition of polyglycosylceramides from human sources.A mixture of gangliosides isolated from human neutrophils was shown to contain the sialic acid binding epitope expressed only by solid grown bacteria. The recognized sequence was identified by a new approach of relating structural analysis and binding activity in mixtures of gangliosides. The ganglioside mixture was separated on TLC and blotted to membranes. The composition and saccharide sequence of up to seven-sugar gangliosides were obtained by fast atom bombardment mass spectrometry after direct desorption from the membrane. The sialic acid linkage position was analysed by a membrane binding assay u s i n g t h e N e u A c a*3 - s p e c i f i c l e c t i n f r o m M a a c k i a a m u r e n s i s a n d t h e N e u A c a*6 - s p e c i f i c l e c t i n f r o m S a m b u c u s n i g r a . T h e s i a l i c a c i d l i n k a g e p o s i t i o n i n t w o s e p a r a t e b a n d s o f t h e f i v e - s u g a r i n t e r v a l w a s a l s o a n a l y s e d b y a n o v e l t e c h n i q u e o f t r i f l u o r o a c e t o l y s i s followed by gas chromatography-mass spectrometry. The upper band contained 3-linked sialic acid and was recognized by H. pylori, while the lower band, which was not recognized, contained 6-linked sialic acid.The second sialic acid-containing epitope restricted to polyglycosylceramides fom human erythrocytes (blood group O) was partially characterized. A new way of molecular fingerprinting of these highly complex and microheterogeneous polyglycosylceramides was adopted based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In a separate study, polyglycosylceramides were peracetylated and subfractioned by anion exchange chromatography. Three subfractions were deacetylated, analysed by MALDI-TOF MS and 1H NMR spectroscopy. Two-dimensional 1H TOCSY-spectra of the mixtures showed that the first fraction contained 3-linked sialic acid and the second and third fractions contained both 3- and 6-linked sialic acid. Only neutral species were present in the first acetylated fraction since it was eluted with solvent lacking salt. The presence of sialic acid in this fraction was explained by a selective lactonization under the conditions used for peracetylation. H. pylori recognized all three fractions, indicating that 3-linked rather than 6-linked sialic acid is essential for binding. .