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Investigating the CEACAM1 Adhesive Properties of Moraxella Catarrhalis Identifying a Novel CEACAM1 Adhesion Protein Found in Mx13l

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Investigating the CEACAM1 Adhesive Properties of Moraxella Catarrhalis Identifying a Novel CEACAM1 Adhesion Protein Found in Mx13l This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Mee, Jodie F Title: Investigating the CEACAM1 adhesive properties of Moraxella catarrhalis Identifying a novel CEACAM1 adhesion protein found in Mx13L 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. 2020 INVESTIGATING THE CEACAM1 ADHESIVE PROPERTIES OF MORAXELLA CATARRHALIS IDENTIFYING A NOVEL CEACAM1 ADHESION PROTEIN FOUND IN MX13L JODIE MEE A dissertation submitted to the University of Bristol in accordance with the requirements for the award of the degree of MSc by Research in Cellular and Molecular Medicine in The Faculty of Life Sciences . School- Cellular and Molecular Medicine Word Count: 17498 1 ABSTRACT Moraxella catarrhalis is an upper respiratory pathogen often implicated as the cause otitis media infections, as well as exacerbating chronic obstructive pulmonary disease. One of the human receptors identified for Moraxella catarrhalis is Carcinoembryonic antigen-related cell adhesion molecule 1 (CC1), normally the surface adhesins UspA1 and UspA2V use the CC1 specific binding sequence within the previously identified rD-7region of UspA1 to interact with CC1. ATCC25240 is a strain of Moraxella catarrhalis which lacks the rD-7 region within its UspA1. This study revealed that a variant of ATCC25240 dubbed MX13L, which also lacks the rD-7 regions binds to CC1. The unknown adhesin present in MX13L interacted with CC1 in a heat modifiable manner. Using a combination of bioinformatics and the ez tn5 transposon to generate a transposon library of MX13L in conjunction with immune overlay assays, we identified that WP_003661028 (BOMP 8) as a putative candidate protein for the novel adhesin. i 2 ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr Darryl Hill for providing me the opportunity to do this MRes as well as his support throughout. I would also like to thank Matt for putting up with me and helping me with my early lab work. Thanks to my undergraduate students Olivia and Anastasia, who performed many of the repeats required to validate my experiments. I’d like to thank the rest of the Hill group for fish and chip Fridays, which made long experiments much more palatable. Thanks to everyone else in C73 for being great office mates. I would like to thank my parents for providing me with the funding required to complete this endeavour as well as their unwavering support. Lastly, I would like to thank Ollie for dealing with my various crises and providing snacks on demand whilst I was writing my thesis. ii 3 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 1 Abstract ................................................................................................................ i 2 Acknowledgements .............................................................................................. ii 3 Author’s declaration ............................................................................................ iii 4 Table of Figures .................................................................................................. ix 5 Abbreviations ...................................................................................................... xi 1 Introduction ......................................................................................................... 1 Background information ................................................................................ 1 Taxonomy ..................................................................................................... 1 Epidemiology ................................................................................................. 4 1.3.1 Diseases in children ............................................................................... 4 1.3.2 Diseases in adults .................................................................................. 5 1.3.3 Septicaemia and bacteraemia ................................................................ 5 Antibiotic resistance ...................................................................................... 6 Vaccine development .................................................................................... 7 1.5.1 Challenges .............................................................................................. 7 1.5.2 Benefits ................................................................................................... 9 Adhesion mechanisms .................................................................................. 9 1.6.2 Trimeric autotransporters ...................................................................... 12 iv CEACAM ..................................................................................................... 19 1.7.1 Carcinoembryonic antigen family .......................................................... 19 1.7.2 CEACAM1 ............................................................................................ 20 2 Aims .................................................................................................................. 23 Value ........................................................................................................... 23 Structure ..................................................................................................... 24 Objectives ................................................................................................... 24 3 Methods ............................................................................................................ 25 Cell strains and growth conditions ............................................................... 25 3.1.1 Bacterial strains and growth conditions ................................................ 25 3.1.2 Eukaryotic strains and growth conditions.............................................. 26 3.1.3 Freezing eukaryotic cells ...................................................................... 27 Producing recombinant CC1-Fc .................................................................. 27 3.2.1 Creating a stable CC1-Fc secreting cell line ......................................... 27 3.2.2 Determining the fidelity of CC1-Fc ........................................................ 34 Identifying a novel CC1 binding protein ....................................................... 36 3.3.1 Assessing MX13L interactions with CC1 .............................................. 36 3.3.2 Identifying target proteins ..................................................................... 39 v 3.3.3 Transposon library ................................................................................ 46 4 Results – Stable CC1 secreting cell line ........................................................... 50 Creating a stable CC1-Fc secreting cell line ............................................... 50 Optimising the zeocin concentration required to select for successful transfection ........................................................................................................... 50 Amplifying the CC1-Fc gene from the full length CC1 gene ........................ 51 4.3.1 Cloning of CC1-Fc PCR product into XL10 Gold cells .......................... 54 Assessing for CC1 production in transfected cells. ..................................... 57 Assessing the production of CC1-Fc by monoclonal populations of transfected CHO cells. ............................................................................................................ 59 Determining the concentration of CC1-Fc ................................................... 61 Comparing the rD-7 binding of CC1-Fc and oCC1-Fc. ................................ 63 SDS-Page gel and western blot to determine the purity of CC1-Fc. ........... 65 Conclusions................................................................................................
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