Exploring Fructan Utilisation by Members of the Human Intestinal Microbiota Sarah A. Shapiro A Thesis Submitted for the Degree of Doctor of Philosophy 2012 – 2015 Institute for Cell and Molecular Biosciences Newcastle University i ii Acknowledgements Foremost, I would like to acknowledge my supervisor, Dr. David Bolam. Dave has entertained a barrage of unanswerable questions and somewhat unconventional experimental designs with boundless patience. Without his guidance, expertise, patience and support, this thesis would not have been possible. I would like to extend my thanks to Professor Harry Gilbert, who has provided me with invaluable advice, support and encouragement throughout my project. I am extremely grateful to all of my colleagues, for their advice, training and friendship. I would also like to acknowledge my collaborators who have contributed to my research; Mr. Carl Morland who provided excellent technical support throughout my work, Professor Glenn Gibson for allowing me access and training with his facilities within the University of Reading and Dr. Arnaud Baslé with whom I have collaborated extensively to obtain x-ray crystal structures. To my Fiancé, Jonathan Ward, I am forever grateful for all the unwavering support and encouragement over the years. i ii Abstract The human gut microbiota contributes to host health and wellbeing in numerous ways, including through polysaccharide fermentation and the production of beneficial short chain fatty acids. Microbiota accessible carbohydrates (MACs) pass through the early digestive tract intact to nourish the microbiota. Fructans are plant derived fructose polymers found in the diet which act as MACs and elevate short chain fatty acid production. Two linkage types are known between fructose units within fructans, β2-1 and β2-6; homopolymers of these are inulin and levan respectively. Fructan structures containing both β2-1 and β2-6 linkages are common within the human diet, but the role of these as MACs have not been studied. Fructan, particularly inulin and fructo- oligosaccharides (FOS) have been used as prebiotics to selectively support beneficial members of the microbiota. Despite widespread use of inulin as a prebiotic there is a paucity of data regarding the mechanisms employed by the microbiota to recognise, import and degrade this glycan. In this thesis, several plant derived fructan extracts were visualised using high performance anion exchange chromatography and thin layer chromatography to confirm these contained non-linear structures. We show that these non-linear structures may support both inulin- and levan- utilising Bacteroides species, despite previously identified mutually exclusivity for one of the two homopolymers. Two key inulin using species were chosen as models to probe inulin utilisation within the microbiota; the Gram negative Bacteroides ovatus , and the Gram positive Bifidobacterium adolescentis . B. ovatus is a prominent member of the healthy human microbiota, and contains a large number of polysaccharide utilisation loci within the genome, each tightly regulated and specific for a selected glycan. Here we show that B. ovatus encodes an inulin utilisation system which includes an endo-acting surface located glycoside hydrolase family 91 enzyme comprised of two gene products (BACOVA_04502 and BACOVA_04503). This enzyme has an appended Carbohydrate Binding Module (CBM) which recognises inulin. SusC/D- homologue pairs are carbohydrate binding and import proteins commonly found within the Bacteroidetes phylum. The structure of a SusD-homologue, BACOVA_04504 was solved and we demonstrate that this protein recognises sucrose terminated FOS and inulin, an unusual specificity which likely assists in the rapid import of desirable short chain FOS though a SusC- homologue. High molecular weight inulin is imported through a SusC-homologue for periplasmic degradation by a glycoside hydrolase family 32 enzyme (GH32). A waste sugar, di-fructose anhydride (DFA), is produced by this system from inulin and released into the intestinal environment; we demonstrate that DFA is produced from inulin by faecal microbiota from three healthy humans and that DFA is not subsequently broken down. B. adolescentis is able to rapidly utilise inulin polysaccharide, an unusual phenotype amongst Bifidobacterium , which generally utilise only short chain FOS, Indeed it has been shown that B. adolescentis is supported well during prebiotic treatment. We demonstrate that B. adolescentis contains an additional locus compared with other Bifidobacterium species and that this locus is responsible for inulin utilisation. The locus includes an inulin recognising extracellular solute binding protein which undergoes a significant conformational change upon ligand recognition which we show through structural studies. The locus contains a LacI- homologue which is able to recognise fructan through the periplasmic binding domain, likely to up-regulate the system; and inulin is finally processed internally by a GH32. We explore the niches occupied by each species in the intestine, and predict that B. ovatus “shares” glycan with other microbiota, including members of the bifidobacterium genus. Our data enable deeper understanding of how fructans interact with the intestinal microbiota, potentially underpinning research into novel and personalised prebiotic therapies. iii Contents Acknowledgements i Abstract iii Contents iv List of Figures xi List of Tables xvi Glossary of Terms and Abbreviations xvii Chapter 1. Introduction 1 1.1. Exploring the Healthy Human Gut Microbiota ....................................1 1.1.1. Introduction ........................................................................................1 1.1.2. Production of Short Chain Fatty Acids and Other Metabolites .............4 1.1.3. Diversity of the Human Gut Microbiota ..............................................8 1.1.4. The Infant Microbiota ....................................................................... 10 1.1.5. The Microbiota and Disease .............................................................. 11 1.2. Dietary Fibres, MACs and the Human Microbiota ............................ 12 1.2.1. Dietary Fibre and Microbiota Accessible Carbohydrates (MACs) ..... 12 1.2.2. The Concept of Prebiotics ................................................................. 16 1.3. Fructans ............................................................................................ 18 1.3.1. Structure and Function ...................................................................... 18 1.3.2. Dietary Prevalence ............................................................................ 22 1.3.3. Fructans and Industry ........................................................................ 23 1.4. Carbohydrate and Fructan Active Enzymes ....................................... 24 1.4.1. Carbohydrate Active Enzymes (CAZymes) ....................................... 24 1.4.2. Fructan Active Enzymes: Glycoside Hydrolase Families 32, 68 ................. and 91 ............................................................................................... 27 1.5. Fructan Binding Proteins................................................................... 32 1.6. Carbohydrate Capture and Utilisation by the Gut Microbiota ............ 35 1.6.1. The Distribution of CAZymes Within the Human Microbiota ........... 35 1.6.2. Carbohydrate Harvest by Members of the Bacteroidetes phylum ....... 37 1.6.3. Carbohydrate Harvest by Members of the Firmicutes and ................... Other Phyla ....................................................................................... 39 1.7. Fructan Degradation by the Microbiota ............................................. 41 1.7.1. Fructan Utilisation Loci .................................................................... 41 1.7.2. Other Fructan Utilisation Systems ..................................................... 44 iv 1.8. Research Objectives .......................................................................... 45 Chapter 2. Materials and Methods 46 2.1. Chemicals, Commercial Kits and Water ............................................ 46 2.2. Sterilisation ....................................................................................... 46 2.3. Storage Practices ............................................................................... 46 2.3.1. DNA ................................................................................................. 46 2.3.2. Protein .............................................................................................. 47 2.3.3. Bacterial Cultures ............................................................................. 47 2.3.4. Carbohydrates ................................................................................... 47 2.4. Vectors ............................................................................................. 47 2.5. Bacterial Strains ................................................................................ 48 2.6. Growth Media ................................................................................... 51 2.7. Routine Equipment Usage ................................................................. 54 2.7.1. Centrifugation ................................................................................... 54 2.7.2. Incubators, Heat Blocks and Water Baths .......................................... 54 2.8. Transformation of E.coli ..................................................................