Methodologies for screening of bacteria–carbohydrate interactions: Anti-adhesive milk oligosaccharides as a case study

• Published in: Journal of microbiological methods Vol. 90; no. 1; pp. 53 - 59
• Main Authors: Lane, Jonathan A., Mariño, Karina, Rudd, Pauline M., Carrington, Stephen D., Slattery, Helen, Hickey, Rita M.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.07.2012; Elsevier
• Subjects: adhesion; Animals; anion exchange chromatography; bacteria; Bacterial Adhesion - drug effects; Bacterial–carbohydrate interactions; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Bovine colostrum oligosaccharides; Carbohydrates - chemistry; case studies; Cattle; Chromatography, Liquid - methods; Colostrum - chemistry; cow colostrum; cows; digestion; Escherichia coli; Escherichia coli - drug effects; Escherichia coli - physiology; Escherichia coli Infections - metabolism; Escherichia coli Infections - microbiology; Fundamental and applied biological sciences. Psychology; HILIC chromatography; Holstein; HPAEC chromatography; HT29 Cells; human cell lines; Humans; hydrophilic interaction chromatography; Hydrophobic and Hydrophilic Interactions; Microbiology; milk; Milk - chemistry; oligosaccharides; Oligosaccharides - chemistry; Oligosaccharides - pharmacology; rapid methods; screening; Tandem Mass Spectrometry - methods; Bovine colostrum oligosaccharides; HILIC chromatography; Bacterial–carbohydrate interactions; Escherichia coli; HPAEC chromatography; Bacterial-carbohydrate interactions; Bovine; Chromatography; Case study; Vertebrata; Mammalia; Bacteria; Carbohydrate; Artiodactyla; Veterinary; Ungulata; Enterobacteriaceae; Milk
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2012.03.017

Many studies have demonstrated the capacity of glycan-based compounds to disrupt microbial binding to mucosal epithelia. Therefore, oligosaccharides have potential application in the prevention of certain bacterial diseases. However, current screening methods for the identification of anti-adhesive oligosaccharides have limitations: they are time-consuming and require large amounts of oligosaccharides. There is a need to develop analytical techniques which can quickly screen for, and structurally define, anti-adhesive oligosaccharides prior to using human cell line models of infection. Considering this, we have developed a rapid method for screening complex oligosaccharide mixtures for potential anti-adhesive activity against bacteria. Our approach involves the use of whole bacterial cells to "deplete" free oligosaccharides from solution. As a case study, the free oligosaccharides from the colostrum of Holstein Friesian cows were screened for interactions with whole Escherichia coli cells. Reductions in oligosaccharide concentrations were determined by High pH Anion Exchange Chromatography and Hydrophilic Interaction Liquid Chromatography (HILIC–HPLC). Oligosaccharide structures were confirmed by a combination of HILIC–HPLC, exoglycosidase digestion and off-line negative ion mode MS/MS. The depletion assay confirmed selective bacterial interaction with certain bovine oligosaccharides which in previous studies, by other methodologies, had been shown to interact with E. coli. In particular, the bacterial cells depleted the following oligosaccharides in a population dependent manner: 3′-sialyllactose, disialyllactose, and 6′-sialyllactosamine. The assay methodology was further validated by studies in which we demonstrated the inhibitory activity of 3′-sialyllactose, and a mixture of bovine colostrum oligosaccharides, on E. coli adhesion to differentiated HT-29 cells. ► Methodology to screen glycans for potential anti-infective activity against bacteria. ► Successfully validated the proposed methodology by inhibition studies with HT-29 cells. ► Identified 37 potential anti-infective glycans in bovine colostrum. ► Demonstrated E. coli interactions with 3′-sialyllactose, disialyllactose, and sialyllactosamine. ► Colostral oligosaccharides and 3′-sialyllactose prevent E. coli adhesion to HT-29 cells.