Helicobacter pylori Growth Assay for Glycans
Helicobacter pylori (H. pylori) is a Gram-negative microaerophilic bacterium isolated from the gastric mucosa of chronic gastritis patients (Warren and Marchall 1983). It is now well established that this microbe is associated with pathogenesis of gastric disorders such as chronic active gastritis, peptic ulcer, gastric cancer, and malignant lymphoma of the MALT type. Although H. pylori infects about half the world’s population, only a fraction of infected patients develop severe gastric disease, suggesting the presence of a defense mechanism in the gastric mucosa itself. H. pylori colonizes surface mucous cells of the gastric mucosa and is rarely found in gland mucous cells such as mucous neck cells and pyloric gland cells located in deeper portions of the mucosa (Hidaka et al. 2001). O-glycans with terminal α1,4-linked GlcNAc residues [GlcNAcα1 → 4Galβ1 → 4GlcNAcβ1 → 6(GlcNAcα1 → 4Galβ1 → 3)GalNAcα → Ser/Thr] are unique to gland mucins secreted from gland mucous cells (Ishihara et al. 1996). Recently, we showed that α1,4-GlcNAc-capped O-glycans suppress H. pylori growth (Kawakubo et al. 2004). We further demonstrated that cholesterol-α-d-glucopyranoside (CGL), a major cell wall component of Helicobacter species, is important for H. pylori survival and that CGL biosynthesis was inhibited by exogenous α1,4-GlcNAc-capped O-glycans. More recently, we have successfully cloned cholesterol α-glucosyltransferase (CHLαGcT) from H. pylori, which is responsible for the CGL biosynthesis, and demonstrated that enzymatic activity of CHLαGcT is suppressed by a synthetic α1,4-GlcNAc-capped O-glycan (Lee et al. 2006). Here, I describe the procedure for a bacterial growth assay to test the effects of glycans on H. pylori growth.
KeywordsGastric Mucosa Chronic Active Gastritis Mucous Neck Cell Pylorus Growth Major Cell Wall Component
Unable to display preview. Download preview PDF.
- Ishihara K, Kurihara M, Goso Y, Urata T, Ota H, Katsuyama T, Hotta K (1996) Peripheral α-linked N-acetylglucosamine on the carbohydrate moiety of mucin derived from mammalian gastric gland mucous cells: epitope recognized by a newly characterized monoclonal antibody. Biochem J 318:409–416PubMedGoogle Scholar
- Lee H, Kobayashi M, Wang P, Nakayama J, Seeberger PH, Fukuda M (2006) Expression cloning of cholesterol α-glucosyltransferase, a unique enzyme that can be inhibited by natural antibiotic gastric mucin O-glycans, from Helicobacter pylori. Biochem Biophys Res Commun 349:1235–1241PubMedCrossRefGoogle Scholar
- Warren JR, Marchall BJ (1983) Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1:1273–1275Google Scholar