This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Brewer, Matt L Title: Investigating Fusobacterium pathogenesis using molecular and genomic methods to inform vaccine design General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. Investigating Fusobacterium pathogenesis using molecular and genomic methods to inform vaccine design Matthew L. Brewer A dissertation submitted to the University of Bristol in accordance with the requirements for award of the degree of Doctor of Philosophy (PhD) in the Faculty of Life Sciences School of Biochemistry, September 2018 Word Count: 38419 Abstract Abstract Cellular adhesion is crucial in the pathogenesis of many bacterial species, where receptor- ligand interactions mediate host colonisation and/or invasion leading to host pathologies. Fusobacterium nucleatum (Fn) is a bacterial species gaining greater interest due to recent links with a variety of diseases, such as colorectal cancer. Fn is known to harbour a vast array of adhesive proteins (adhesins), however only a few have been studied in depth, such as FadA. This study aims to characterise a new previously unstudied subset of trimeric autotransporter adhesins (TAAs) found within Fn, responsible for binding the human receptor CEACAM1. These proteins were given the name CEACAM-binding proteins of Fusobacterium (CbpF). To examine the distribution of these receptors among Fusobacterium spp., we screened and sequenced a library of clinical isolates. From sequencing these strains, we identified two novel species of Fusobacterium (F. oralis sp. nov. and F. ovarium sp. nov.), both of which harboured CbpF. While performing the taxonomic analyses on the new strains we addressed the conflicting nomenclature and phylogenetic boundaries with respect to the genus. By utilising computational methods, we could confidently delineate species and show how the genus should be organised to better reflect the genomic differences and similarities between strains. Through screening two different types of CbpF from different species we confirmed the ability of both classes to bind CEACAM1 through proteomic- and cellular adhesion-based assays as well as showing that both classes of protein were capable of binding to CEA (CEACAM5), but not to other CEACAM variants examined. This highlighted the highly specific nature of these proteins, which was explored further by examining point mutants of CEACAM1, of which few showed any significant adhesion. As well as examining CbpF, we briefly looked at two other TAAs from Fn: FN0471 and FN0735; the former of which could bind indiscriminately to HeLa cells, thus indicating another important adhesin yet to be fully characterised. Structural analysis of CbpFs highlighted a gap in the literature with respect to TAA motifs and topologies, where no known structures showed significant homology to large portions of the proteins particularly in a region predicted to be occupied with a coiled-coil motif. X-ray crystallography, SAXS and CD were used to infer structural features of CbpF, however an atomic resolution structure could not be accurately produced from a protein crystal X-ray diffraction dataset. The work conducted here lays the foundation for additional studies into TAAs from Fusobacterium highlighting the requirement for increased detail on how these proteins contribute to pathogenesis and whether these proteins could be used as potential future vaccine candidates. i Acknowledgements Acknowledgements I would like to thank my supervisors, Dr Darryl Hill and Prof Leo Brady for providing me the opportunity to do this PhD as well as their unwavering support throughout its duration. Additionally, I would like to thank the SWBio DTP for accepting me and putting together a great doctoral training course. I also have to thank my progression panel, Ross and Paul, for providing additional support and helping to keep me on track throughout. Secondly, I have to say a huge thank you to all the many people over the last four years that have taught me the vast array of techniques I used in this work. Firstly, to Clio for helping me massively in my early stages in the lab, as well as providing me with the wisdom of a newbie post-doc. Other members of the Hill and Brady groups that deserve my appreciation are Ahmad and Storm for showing me the ways of protein purification and to Darryl and Nibras with tissue culture and cell imaging etc. Honourable mentions go to Nick, Ash, Johnny, Sesh and various DLS staff for all their help with SAXS, CD, MD and X-ray crystallography. In addition to the practical and academic support, I would like to thank our wonderful C-floor tech team, including Doug, Dave, Andy, Bill and Anne for all the general lab equipment support as well as making some of the less desirable buffers and media! Especially Anne for short notice media orders. I also want to thank my office pals including Henry, Alex and various other people, including the Finn group, that have had to put up with my (and Henry’s) antics over these past years. Without the relaxed working environment, I don’t think I would have stayed sane over the course of my PhD. Lastly, I would like to thank Bronwyn, my family and various other friends for being there to listen to me waffle on endlessly about a topic they clearly have no interest in and to give me a chance to not have to think about science. ii Author’s Declaration Author’s Declaration I declare that the work in this dissertation was carried out in accordance with the requirements of the University's Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate's own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. Signed: .................................... Date: ........................................ iii Contents Contents Abstract i Acknowledgements ii Author’s Declaration iii Contents iv List of Figures x List of Tables xiii Acronyms and Abbreviations xiv Chapter 1: Introduction 1 1.1 The Fusobacterium genus 1 1.1.1 Biophysical and Biochemical Characteristics 1 1.1.2 Taxonomy 2 1.1.3 Health and Disease 4 1.1.3.1 Periodontal Disease 4 1.1.3.2 Colorectal cancer 6 1.1.3.3 Preterm births 7 1.1.3.4 Lemierre’s Syndrome 7 1.1.3.5 Footrot 8 1.1.3.6 Other Diseases 9 1.1.3.7 Treatment 9 1.1.4 Virulence Factors 9 1.1.4.1 Adhesive Proteins 10 1.2 Trimeric Autotransporter Adhesins 11 iv Contents 1.2.1 Structure and Assembly 11 1.2.2 Specialised folds 13 1.2.3 Receptor binding 18 1.3 CEACAMs 19 1.3.1 Functions 19 1.3.2 Structural Properties 22 1.3.3 Pathogen Interplay and Disease 26 1.4 Fusobacterium Vaccine Antigen Targets 27 1.5 Aims 28 Chapter 2: Materials and Methods 28 2.1 Bacterial strains and growth conditions 28 2.2 Eukaryotic strains and growth conditions 30 2.3 Gene Cloning 31 2.3.1 Creating a Plasmid for Producing Soluble Recombinant Protein 31 2.3.2 Creating a Plasmid for Surface Expression of Protein 33 2.3.3 Site-directed Mutagenesis 33 2.3.4 Plasmids and Primers 35 2.4 Bacterial Protein Expression and Purification 39 2.4.1 Small-scale Expression and Native Purification 39 2.4.2 Large-scale Expression and Native Purification 40 2.4.3 Large-scale Expression and Denaturing Purification 41 2.4.4 Surface Expression 41 2.5 Fusobacterium Lysate Preparation 41 v Contents 2.6 Human CEACAM IgG1-FC fusion protein production 42 2.6.1 DNA Preparation 42 2.6.2 Large Scale Transient Transfection 42 2.6.3 Small-scale Transfections and Purification 43 2.7 Western blots, Immunodot blots and ELISAs 43 2.7.1 Quantitative ELISA 44 2.8 Adhesion assays 45 2.8.1 Eukaryotic cell preparation 45 2.8.2 Inhibitor preparation 45 2.8.3 Bacterial preparation 45 2.8.4 Cell fixation 46 2.8.5 Cell staining 46 2.8.6 Cell imaging 47 2.9 Crystallography 47 2.9.1 Crystal Looping and Data Collection 48 2.10 Small-angle X-ray Scattering 48 2.11 Molecular Dynamics 49 2.12 Whole Genome Sequencing 49 2.13 Phylogenetics 50 2.13.1 Genome and Proteome Mining 50 2.13.2 Average Nucleotide Identity 50 2.13.3 Maximal Unique Matches and MUMi 50 2.13.4 Pan-Locus Sequence Analysis 51 vi Contents 2.13.5 Phylogenetic Trees 52 2.13.6 Hive Plots 53 2.14 Statistics 53 Chapter 3: A comprehensive phylogenetic analysis of the Fusobacterium genus 54 3.1 Introduction 54 3.2 Whole-Genome Sequencing of Clinical Strains 56 3.3 Method Comparison 59 3.3.1 Pre-screening for Non-Fusobacterium Strains 59 3.3.2 Average Nucleotide Identity 59 3.3.3 MUMmer and MUMi 60 3.3.4 Development of Pan-Locus Sequence Analysis 61 3.3.5 Score Comparison 62 3.4 Defining the Taxonomic Boundary 64 3.5 Reclassification of Multiple Species 67 3.5.1 Identification and Classification of F.