Investigating the molecular mechanisms of the interactions between Lactobacillus reuteri strains and intestinal mucus Faye Jeffers Thesis submitted for the degree of Doctor of Philosophy To The University of East Anglia Institute of Food Research Norwich Research Park Colney Lane Norwich NR4 7UA September 2012 This copy of the thesis has been supplied on the condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. 1 Abstract Mucus is the first point of contact between the gut microbiota and the host. Here we used the gut symbiont Lactobacillus reuteri to investigate the molecular mechanisms underlying the interactions between gut bacteria and mucus. Firstly, the mucus binding ability of a collection of L. reuteri strains from different vertebrate hosts was assessed in vitro against mucus extracted from mouse and porcine gastrointestinal tracts. The adhesion profile was strain-specific showing the highest binding phenotype for strain ATCC 53608 (a pig isolate) and binding ability for a number of L. reuteri human isolates. Genome sequencing of the ATCC 53608 strain and comparative genomics was carried out to gain novel insights in the strain-specific determinants of L. reuteri adhesion to mucus. The second part of this work investigated the occurrence at the genetic and protein level of two specific cell surface proteins, MUB and Lar_0958, expressed by the ATCC 53608 strain and human strains, respectively and their role in mediating mucus binding and autoaggregation ability of L. reuteri strains, as determined by flow-cytometry and in vitro mucus binding assays with recombinant and native proteins. Finally a quantitative proteomic approach, stable isotope labelling with amino acids in cell culture (SILAC), was used to identify novel mucus binding protein candidates. This was achieved through detection of increased cell surface protein expression when L. reuteri was grown in presence of mucins, resulting in induced mucus binding phenotype. Collectively, these results shed new light on the nature and distribution of strain-specific surface proteins mediating L. reuteri adhesion to mucus. 2 Contents Abstract ..........................................................................................................................2 Contents .........................................................................................................................3 List of Figures ..................................................................................................................5 List of Tables ...................................................................................................................7 Preface ...........................................................................................................................8 Abbreviations .................................................................................................................9 Acknowledgements ....................................................................................................... 11 1 Introduction .......................................................................................................... 12 1.1 Physiology of the Vertebrate Gastrointestinal (GI) Tract ......................................12 1.2 Mammalian GI Mucus ...........................................................................................15 1.3 Human Gut Microbiota ..........................................................................................21 1.3.1 Establishment of the microbial community ..................................................21 1.3.2 Composition and ecology of the human gut microbiota ..............................22 1.3.3 Lactobacillus reuteri in the vertebrate GI tract .............................................24 1.4 Bacterial mechanisms of adhesion ........................................................................28 1.4.1 Biofilm formation, aggregation and mucosal recognition ............................28 1.4.2 Bacterial cell surface structures ....................................................................30 1.5 Specific interactions between lactobacilli and mucus ...........................................33 1.6 Aims and objectives of the research project .........................................................40 1.6.1 Aim ................................................................................................................40 1.6.2 Objectives ......................................................................................................40 2 Materials and Methods ......................................................................................... 41 2.1 General Materials ..................................................................................................41 2.2 Microbiology ..........................................................................................................42 2.2.1 Bacterial strains, media and culture conditions ............................................42 2.2.2 Carboxyfluorescein (cF) labelling of bacteria ................................................42 2.3 Biochemistry ..........................................................................................................44 2.3.1 Mucus preparation ........................................................................................44 2.3.2 Mucus characterisation: agarose/polyacrylamide composite gel electrophoresis (AgPAGE) and Western blot ................................................44 2.3.3 Adhesion (Bacterial, Protein and Induction assays) ......................................45 2.3.4 Spectrophotometry autoaggregation assay ..................................................48 2.3.5 Statistical Analysis .........................................................................................48 2.3.6 Flow cytometry (FCM) ...................................................................................48 2.3.7 Immunogold scanning electron microscopy (SEM) .......................................49 2.4 Molecular Biology ..................................................................................................49 2.4.1 Genomic DNA extraction ...............................................................................49 2.4.2 General bioinformatics tools .........................................................................50 2.4.3 Polymerase chain reaction (PCR) of mub and lar_0958 genes .....................50 2.4.4 Agarose gel electrophoresis ..........................................................................50 2.4.5 Random amplified polymorphic DNA (RAPD) analysis ..................................51 2.5 Proteomics .............................................................................................................51 2.5.1 Protein Quantitation Assays ..........................................................................51 2.5.2 Preparation of cell wall extracts, soluble cytoplasmic extracts and spent media extracts ..........................................................................................................52 2.5.3 SDS-PAGE and Western Blotting ...................................................................52 2.5.4 Cell surface proteome extraction ..................................................................53 2.5.5 Purification of MUB and Lar_0958 proteins ..................................................54 3 2.5.6 Preparation of stable isotope labelling with amino acids in cell culture (SILAC) samples..........................................................................................................54 2.5.7 Mass spectrometry of SILAC samples and data analysis ...............................56 3 L. reuteri adhesion to mucus ................................................................................. 58 3.1 Extraction and characterisation of GI mucus ........................................................58 3.2 Development, optimisation and standardisation of the mucus adhesion assay ..62 3.2.1 Saturation of mucus coating and mucus preparation comparison ...............62 3.2.2 Adhesion assay optimisation and standardisation; bacterial cell numbers, viability, labelling and growth phase .............................................................64 3.3 Mucus adhesion ability of L. reuteri strains ...........................................................68 3.4 Role of autoaggregation in mucus binding ............................................................73 3.5 Genome sequencing of L. reuteri ATCC 53608 ......................................................75 3.6 Discussion ..............................................................................................................79 4 Role of MUB and Lar_0958 in L. reuteri adhesion to mucus .................................... 83 4.1 Occurrence of mub genes and proteins in L. reuteri strains .................................84 4.2 Comparison of spontaneous mutant 1063N to wild type ATCC 53608 .................91 4.3 MUB contribution to bacterial cell adhesion to mucus .........................................95 4.4 Adhesion of MUB protein to mucus ......................................................................97 4.5 Occurrence of lar_0958 homologue genes and proteins in L. reuteri strains .....100 4.6 Lar_0958