Human Milk Oligosaccharides: Bioactive Sugars to Exploit the Bacterial Sweet Tooth

Abstract

Carbohydrates, or glycans, are ubiquitous in nature and present across all kingdoms of life - bacteria, fungi, viruses, yeast, plants, animals, and humans. They are essential to many biological processes. Fucose is a deoxyhexose; an unusual sugar, which is abundant in host mucosal surfaces, where it decorates terminal positions of mucin carbohydrate ligands; microbial cell surface structures and is found in some dietary sources [1]. Fucosylated glycans are involved in a variety of physiological processes including immunity, brain development, cancer and host-microbe interactions. As such, opportunities for the detection, prevention, and intervention of bacterial infection through diet or novel therapeutics are possible due the cell surface, species-specific carbohydrates and carbohydrate-binding proteins presented by pathogens. Human milk oligosaccharides (HMOs) are associated with a range of diverse health benefits, acting as prebiotics for commensal bacteria, receptor decoys for pathogens and as immune stimulants. Their bioactive properties provide a unique opportunity to impact bacteria-associated disease in humans and animals. Over 200 different structures of HMO have been identified in human breast milk, significantly more than are present in the milk of livestock and most primates. However, their biosynthesis is largely unknown and there are many challenges that impede either their purification from donated samples or chemical and/or enzymatic synthesis. The aim of this research is to 1) develop improved liquid chromatography strategies for the characterisation and purification of HMOs; 2) investigate the synthesis of fucosylated HMOs and novel, non-natural HMO analogues; 3) assess the ability of HMOs to modulate bacterial growth and 4) develop a selective and sensitive microplate-based assay to detect glycan-bacterial interactions in live bacteria. Liquid chromatography methods were optimised for the characterisation and purification of HMOs, including for the analysis of fucosylation reactions. To enable benchtop analysis and purification of HMOs, a method for the use of HILIC cartridges was developed, which successfully resolved mixtures of HMOs. Investigations with alpha-1,2-fucosyltranferase from H. mustelae, showed that it is a promiscuous enzyme that can accept and convert a variety of di-, tri-, tetra- and pent- saccharides, including core HMO structures and fucosylated glycans and functionalised lactosides. This enzyme is capable of synthesising HMO analogues and the procedure can be scaled up when used in combination with L-fucosekinase/GDP-L-fucose pyrophosphorylase (FKP) in a two-step, one pot reaction. Furthermore, FKP can convert D-Ara and L-Gal into their respective GDP-sugar nucleotides, and these can be processed as donors by alpha-1,2-fucosyltransferase to synthesise non-natural HMO analogues. Microbiological assays revealed that Escherichia coli is able to utilise L-fucose as a sole energy source but not HMOs, nor do HMOs affect microbial growth when co-fed with fucose. Finally, an ELISA-like binding assay was developed which allowed selective binding of E. coli to glycan-functionalised microplates. The methodology can be adapted to gold nanoparticles, quantum dots and gold/graphene chips for use as highly specific biosensors for detection of pathogens or identification of pathogen-specific glycan motifs for the development of targeted drug therapies.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Sabine Flitsch (Supervisor) & Robert Field (Supervisor)