Mucin Utilisation and Host Interactions of the Novel Intestinal Microbe Akkermansia muciniphila Muriel Derrien Promotor Prof. dr. W. M. de Vos Hoogleraar Microbiologie Wageningen Universiteit Co-promotor Dr. E. G. Zoetendal Wetenschappelijk onderzoeker Laboratorium voor Microbiologie Wageningen Universiteit Promotiecomissie Prof. dr. M. R. Müller Wageningen Universiteit Dr. A. C. Ouwehand Danisco Innovation, Kantvik, Finland Dr. H. J. M. Harmsen Rijksuniversiteit Groningen Dr. E. Norin Karolinska Institute, Stockholm, Sweden Dit onderzoek is uitgevoerd binnen de onderzoekschool VLAG Mucin Utilisation and Host Interactions of the Novel Intestinal Microbe Akkermansia muciniphila Muriel Derrien Proefschrift Ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. M.J. Kropff, in het openbaar te verdedigen op dinsdag 15 mei 2007 des namiddags te half twee in de Aula M. Derrien – Mucin utilisation and host interactions of the novel intestinal microbe Akkermansia muciniphila – 2007 PhD. thesis Wageningen University, Wageningen, The Netherlands, with summary in Dutch and French ISBN: 978-90-8504-644-8 Abstract Mucins are the major organic components of the defence barrier, known as mucus, covering epithelial cells in many organs, including the entire gastrointestinal (GI) tract. Microbes that can associate with mucins benefit from this interaction since they can access nutrients. Mucin- degrading bacteria are therefore an important community that have not been extensively studied as the substrate itself, mucin, is a complex and high molecular weight glycoprotein. The work presented here is focused on the identification and isolation of mucin-degrading bacteria from the GI tract, their degradation of mucin and their interaction with the host. Mucin-degrading bacteria were analysed by combining molecular- and cultivation-based approaches. The faecal mucin-degrading bacterial community was found to be highly diverse and host-specific. A novel isolate, representing a novel genus, was cultured from the highest dilution of the enrichment of a single individual. This intestinal isolate, Akkermansia muciniphila, was found to grow to a limited extent on a very limited amount of substrates but grew efficiently on mucin. Phylogenetic analysis based on 16S rRNA sequences indicated that A. muciniphila belonged to the phylum Verrucomicrobia, which was not known to contain intestinal members. Moreover, 16S rRNA genes from A. muciniphila have been retrieved in several clone libraries derived from either faecal or biopsy samples from human and mice. In addition, A. muciniphila was found to be rather abundant in the GI tract. Based on fluorescent in situ hybridisation and quantitative PCR, A. muciniphila was found to represent an average of 109 cells / g faecal sample. A negative correlation between the concentration of faecal mucin and the number of A. muciniphila was observed, suggesting it to be involved in mucin degradation in vivo. Several specific enzymes, mostly glycosidases were found to be secreted during its growth on mucin that was degraded for a major part (85%). Hence, the specific impact of A. muciniphila on the host was investigated in germ-free mice and compared to that of the non mucin-degrading bacterium L. plantarum. Transcriptomic microarray analysis showed that both A. muciniphila and L. plantarum modulated a similar number of genes but that host response was found to be highly specific for each bacterium, depending on the anatomical location. Amongst the major responses, we could detect for A. muciniphila a regulation of the immune response, cell proliferation, cell adhesion and apoptosis, and for L. plantarum a regulation of the lipid metabolism. Overall, this work has brought new insights into the mucin-degrading community of bacteria, and in particular the role of A. muciniphila, an abundant human mucin-degrading bacterium. Keywords. Mucin, A. muciniphila, mucin degradation, molecular techniques, host response Table of contents Preface Chapter 1 General introduction 11 Chapter 2 Prebiotics and other microbial substrates for gut functionality 33 Chapter 3 Monitoring of the bacterial mucin-degrading consortium 43 Chapter 4 Akkermansia muciniphila gen. nov., sp. nov., a human intestinal 53 mucin-degrading bacterium Chapter 5 Analysis the mucin-degrading enzymes of Akkermansia muciniphila 65 Chapter 6 The mucin-degrader, Akkermansia muciniphila, is an important 75 member of the human intestinal tract. Chapter 7 Mucin secretion and fructooligosaccharides in the intestine: 89 the role of the mucin-degrading bacterium Akkermansia muciniphila Chapter 8 Gene profiling of mice responses after mono-association 101 with the intestinal mucin-degrading bacterium Akkermansia muciniphila and the commensal Lactobacillus plantarum Chapter 9 Summary, concluding remarks and future perspectives 131 Appendix Literature cited 139 Nederlandse samenvatting 156 Résumé en français 159 Acknowledgements 165 About the author 169 List of publications 171 Education 173 Preface The work presented here is focused on the identification and isolation of mucin-degrading bacteria from the gastrointestinal tract, their degradation of mucin, and their interaction with the host. Chapter 1 provides an introduction in the structure and properties of mucin, the known bacteria that degrade mucin, and the interactions between bacteria and the host. Chapter 2 is a review on intestinal substrates used by the intestinal microbiota, including prebiotics and substrates secreted by the host including mucus. Chapter 3 describes the monitoring of faecal bacterial communities able to grow on isolated mucin. Fingerprinting of 16S rRNA amplicons by denaturing gradient gel electrophoresis followed by cloning and sequencing of the amplicons, resulted in the identification of the major populations. Chapter 4 details the isolation from human faeces of a novel bacterium, Akkermansia muciniphila, able to grow on mucin as both carbon and nitrogen source, and its subsequent characterisation by molecular, phylogenetic and physiological approaches. Chapter 5 provides an overview of the repertoire of mucinolytic enzymes of A. muciniphila by using a combination of biochemical and physiological approaches. Chapter 6 describes the validation of a specific probe targeting a part of the 16S rRNA gene of A. muciniphila, and its application in faecal samples for the quantification of A. muciniphila by using fluorescent in situ hybridisation combined with flow cytometry. Chapter 7 aims to describe the relation of the number of cells of A. muciniphila and the amount of mucus produced by human volunteers that were subject to the consumption of fructooligosaccharides (FOS). A possible link between the FOS diet, mucin increase and A. muciniphila population dynamics was investigated. Chapter 8 is addressing the impact on the host by A. muciniphila. Germ-free mice were mono-associated with A. muciniphila and the global transcriptional response was determined by high throughput microarrays representing the genome of the mouse. Chapter 9 summarises the work of the thesis and provides perspectives for future research. à ma famille, [ CHAPTER 1 \ General Introduction The human gastro-intestinal (GI) tract is essentially a tube, which starts from the mouth and ends at the anus. The lumen of the GI tract is continuous with the external environment and only separated from the inside by a single layer of intestinal cells that are covered by a mucus layer. The intestinal lumen is colonised since birth by diverse microbial communities – collectively known as the microbiota - that predominantly consist of bacteria. There is a growing interest in the interactions between the microbiota, the host cells and nutrients as they constitute the three major key players of the intestinal ecosystem that are involved in health and disease. Hence, their main attributes are described here with special attention for the microbiota, the intestinal mucins and their interactions. Chapter 1 The human gastrointestinal tract and its microbiota Following food intake, the digestive process starts in the mouth during mastication and maceration, and the food components are then passed on to the stomach for further treatment by acids and digestive enzymes. The last phase of digestion takes place in the small and large intestine (Fig. 1.1). The small intestine, composed of three distinct anatomical and functional parts, duodenum, jejunum and ileum, plays a major role in absorption of nutrients via action of several enzymes converting carbohydrates into monosaccharides, lipids into fatty acids and glycerol, and proteins into amino acids. Part of the food components are metabolised and subsequently absorbed in the small intestine but the complex ones reach the colon in an intact form for further microbial fermentation. The colon is considered as the metabolically most active site from the GI tract. It starts from the caecum, continues to ascending, transverse and descending colon and finishes at the rectum where the luminal contents leave the body as faeces. From the upper part of the GI tract to the lower part, the pH increases gradually in parallel to concentration of bacteria (Fig. 1.1). Chemical and mechanical digestion Stomach pH 2, 45 cm, transit 2-6 h 105 bacteria /g Duodenum Nutrients uptake Jejunum Small intestine pH 5-6, 5-7 m, transit 3-6 h 106-108 bacteria /g Ileum Caecum Water absorption Large intestine Colon pH 6-8, 1.50 m, transit 15-40 h Rectum 108-101010 bacteria /g