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Proteins of Lactobacillus Reuteri

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Proteins of Lactobacillus Reuteri STRUCTURAL AND FUNCTIONAL CHARACTERISATION OF MUCUS ADHESION PROTEINS OF LACTOBACILLUS REUTERI Sabrina Etzold, M.Sc. Thesis submitted for the degree of Doctor of Philosophy To The University of East Anglia Institute of Food Research School of Biology (University of East Anglia) September 2013 © This copy of the thesis has been supplied on 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. Mucus adhesins are thought to be key mediators in the mucus adhesion of commensal Lactobacillus species. However, knowledge on the structural or functional basis of adhesin interaction with mucin glycoproteins, the main component of mucus, is limited. This work describes the biochemical and structural properties of two cell-surface proteins from Lactobacillus reuteri, the mucus-binding protein (MUB) and the Lar0958 protein, and their mucin binding ability. MUB from L. reuteri ATCC 53608 consists of 14 Mub repeats, six type 1 and eight type 2. Single and tandem Mub repeats were heterologously expressed and purified for structural and functional studies. The three-dimensional structure of the Mub type 1 MubRV was determined by X-ray crystallography and revealed two structural domains, B1 and B2. Furthermore, structural homology between MubRV and fibre-like adhesins of Gram-positive pathogens was identified. Small angle X-ray scattering experiments of single and tandem Mub repeats suggested an elongated structure of MUB in a ‘beads on a string’ arrangement. Functional studies of recombinant Mub repeats and the full-length native MUB isolated from Lactobacillus spent culture media, demonstrated binding to different mucins in vitro. Sugar inhibition experiments and glycan arrays suggested the involvement of sugar recognition in MUB protein binding to mucins. Lar0958 is a modular protein of six Lar0958 repeats present on the cell-surface of L. reuteri DSM 20016T. The crystal structure of a single recombinant Lar0958 repeat was solved at 1.5 Å, demonstrating a similar protein fold to Mub repeats. In addition, the Lar0958 repeat shows structural similarity to internalin proteins of the pathogen Listeria monocytogenes. Taken together these results provide new insights into the structural organisation of lactobacilli mucus adhesins and their interaction with mucins, highlighting similarities with Gram-positive adhesins of pathogenic bacteria. 2 ACKNOWLEDGEMENTS I would not have been able to complete the journey of the past four years without the support of numerous people. First and foremost, I would like to express my deep appreciation to my supervisors: Dr. Nathalie Juge, Dr. Andrew Hemmings and Prof. Rob Field. You taught me well and I thank you for all your guidance, support and advice throughout my Ph.D. Especially to Nathalie for your dedication and the time and effort put into this Ph.D. thesis especially during the writing up process. I would like to thank my examiners Prof. James Naismith and Dr. Gary Rowley for giving me the opportunity to discuss my work. I gratefully acknowledge the contribution of my collaborators to this work by providing biological material and technical expertise: Prof. Gunnar Hansson, Prof. Sabine Flitsch, Prof. Lokesh Joshi, Dr. David Bolam, Prof. William Willats and Prof. Michael McGuckin. Many thanks go to the past and present members of the Juge lab: Donald MacKenzie, Louise Tailford, Emmanuelle Crost, Christine Fuell, Faye Jeffers, Olivia Kober and Elizabeth Thursby, and to my friends of the IFR lunch crowd and ‘across the road’ for their help and for making the last four years so enjoyable. To Dagmara, I could not have wished to find a better friend in Norwich, you made it a better place. To my friends back home, Steffi, Steffi and Tanja, thank you for believing in me, not giving up on me and for being there at the other end of the phone. Finally, I would like to express my undying gratitude to my parents for their continuing love, deep trust and immeasurable support, it is thanks to you that I arrived where I am now. Zum Schluss möchte ich meinen Eltern meine unendliche Dankbarkeit ausdrücken für ihre fortdauernde Liebe, ihr tiefes Vertrauen und ihre grenzenlose Unterstützung, ich habe es euch zu verdanken, dass ich heute hier angekommen. 3 TABLE OF CONTENTS ABSTRACT _________________________________________________________________ 2 ACKNOWLEDGEMENTS ______________________________________________________ 3 TABLE OF CONTENTS _______________________________________________________ 4 LIST OF FIGURES ___________________________________________________________ 7 LIST OF TABLES ____________________________________________________________ 9 PREFACE _________________________________________________________________ 10 ABBREVATIONS ___________________________________________________________ 12 CHAPTER 1 INTRODUCTION _________________________________________________ 15 1.1 THE GASTROINTESTINAL (GI) TRACT __________________________________________ 15 1.1.1 PHYSIOLOGY OF THE MAMMALIAN GI TRACT ____________________________________ 15 1.1.2 THE GI MUCUS LAYER ____________________________________________________ 17 1.2 MUCINS IN THE GI TRACT ___________________________________________________ 20 1.2.1 THE MEMBRANE-BOUND MUCIN MUC1/MUC1 __________________________________ 23 1.2.2 THE GEL-FORMING MUCIN MUC2/ MUC2 ______________________________________ 24 1.2.3 MUC2 O-GLYCOSYLATION ________________________________________________ 26 1.3 MECHANISMS OF HOST-MICROBE INTERACTION IN THE GI TRACT ______________________ 28 1.3.1 THE GASTROINTESTINAL MICROBIOTA COMPOSITION ______________________________ 28 1.3.2 BACTERIAL INTERACTION AT THE MUCOSAL SURFACE _____________________________ 31 1.4 BACTERIAL ADHESINS _____________________________________________________ 32 1.4.1 MUCUS ADHESINS IN PATHOGENIC BACTERIA ___________________________________ 35 1.4.2 MUCUS ADHESINS OF LACTOBACILLUS SPECIES _________________________________ 38 1.5 STRUCTURAL BASIS OF BACTERIAL MUCUS ADHESINS ______________________________ 43 1.6 AIMS AND OBJECTIVES OF THIS RESEARCH PROJECT ______________________________ 48 CHAPTER 2 MATERIAL AND METHODS _______________________________________ 49 2.1 MATERIALS _____________________________________________________________ 49 2.1.1 STANDARD BUFFERS _____________________________________________________ 49 2.1.2 SUGARS AND MUCINS ____________________________________________________ 49 2.1.3 LECTINS AND ANTIBODIES _________________________________________________ 50 2.1.4 BACTERIAL STRAINS, MEDIA COMPOSITION AND CULTURE CONDITIONS _________________ 51 2.2 MOLECULAR BIOLOGY _____________________________________________________ 53 2.2.1 POLYMERASE CHAIN REACTION (PCR) _______________________________________ 53 2.2.2 PLASMID DNA PURIFICATION ______________________________________________ 53 2.2.3 DNA AGAROSE GEL ELECTROPHORESIS ______________________________________ 54 2.2.4 DNA CLONING IN EXPRESSION VECTORS ______________________________________ 54 2.2.5 BACTERIAL TRANSFORMATION ______________________________________________ 55 2.2.6 RECOMBINANT DNA ANALYSIS _____________________________________________ 55 2.2.7 ESTIMATION OF DNA CONCENTRATION _______________________________________ 56 2.3 BIOCHEMISTRY __________________________________________________________ 56 4 2.3.1 RECOMBINANT PROTEIN PRODUCTION ________________________________________ 56 2.3.1.1 Protein expression ___________________________________________________ 56 2.3.1.2 Protein extraction ____________________________________________________ 56 2.3.1.3 Protein purification ___________________________________________________ 57 2.3.2 MUB PURIFICATION _____________________________________________________ 58 2.3.3 ESTIMATION OF PROTEIN CONCENTRATION _____________________________________ 58 2.3.4 PROTEIN GEL ELECTROPHORESIS ___________________________________________ 59 2.3.4.1 Polyacrylamide gel electrophoresis (PAGE) ________________________________ 59 2.3.4.2 Agarose-polyacrylamide composite gel electrophoresis (AgPAGE) ______________ 59 2.3.4.3 Isoelectric focusing (IEF) ______________________________________________ 60 2.3.4.4 Staining of gels ______________________________________________________ 60 2.3.4.5 Western blotting _____________________________________________________ 61 2.3.5 PROTEIN DETECTION VIA ANTIBODIES AND LECTINS _______________________________ 61 2.3.6 PROTEIN BINDING ASSAYS _________________________________________________ 62 2.3.6.1 Membrane protein binding assays after electrophoresis ______________________ 62 2.3.6.2 Glycan array ________________________________________________________ 63 2.3.6.3 Mass Spectrometry glycan array ________________________________________ 63 2.3.6.3.1 Functionalisation of Au-surfaces _______________________________________ 63 2.3.6.3.2 Protein binding on functionalised Au-glycan chips _________________________ 64 2.3.6.4 Slot-blot assay _______________________________________________________ 64 2.3.6.5 Microtitre plate assays ________________________________________________ 65 2.4 BIOPHYSICS ____________________________________________________________ 66 2.4.1 ISOTHERMAL TITRATION CALORIMETRY (ITC) ___________________________________ 66 2.4.2 CIRCULAR DICHROISM (CD) _______________________________________________
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