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Functional Genomics of the Insect-Vector Symbiont, Sodalis Glossinidius

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Functional Genomics of the Insect-Vector Symbiont, Sodalis Glossinidius Functional Genomics of the Insect-Vector Symbiont, Sodalis glossinidius Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Lauren Clare Gordon November 2019 Contents ____________________________________________________________________ Contents………………………………………………………………………………………………………………..…I Acknowledgments………………….………………………………………………………………………………IV Abbreviations………………………………………………………………………………………………………….V Abstract………………………………………….……………….…………….………….………....……………….IX 1. Introduction…………………………………………….…….…….……...…………………………………..1 1.1. Vector Epidemiology…………………………………….….…….……..……...….……………….1 1.1.1. Human African Trypanosomiasis…….….….………….…….…………….…………...1 1.1.2. Animal African Trypanosomiasis………….………….………….……………………….3 1.1.3. Parasite Lifecycle in Insect Vector………….…….…….…………………………….….3 1.1.4. Vector Control Strategies…………………….….…….…………………………………….6 1.1.5. Symbiont Involvement in Vector-Parasite Interactions………………………..7 1.2. Sodalis glossinidius……………………………………………………………………………………11 1.2.1. Overview……………………………………………………………………………………………11 1.2.2. Genetics………….….……….…………………………………………………………………….14 1.2.2.1. Annotation History…………..……..………..………….………….……….………14 1.2.2.2. Metabolic Predictions……….………...…….….…….…….….………………….20 1.2.3. Evolutionary Genomics of Symbionts……….….……..……..…..…………………23 1.2.4. Genetic Manipulation for Functional Understanding……….……….…….…26 1.3. Aims and Objectives……….……….…….…..……….……………………………………………31 2. Metabolic Profiling of Sodalis glossinidius………………………………………………..……32 2.1. Introduction…...……….………….…………………………………………………………………..32 2.2. Methods…………………………………………………………………………………………………..36 2.2.1. Minimalistic versus Complex Media…………………………………………………..36 2.2.2. Component-Supplemented Minimal Medium…….………….…….……………36 2.2.3. Carbon Source Screen.………….……………..…..……………………………………….38 2.3. Results……………………………………………………………………………………………………..39 2.3.1. Minimalistic versus Complex Media…………………………………………………..39 2.3.2. Component-Supplemented Minimal Medium…….……….……….……………40 2.3.3. Carbon Source Screen…….……….…….……..…….…………………………………….42 2.4. Discussion………………………………………………………………………………………………..46 3. Method Development for the Transformation of Sodalis glossinidius….…..…..49 3.1. Introduction…...…….………….……………………………………………………………………..49 3.2. Methods…………………………………………………………………………………………………..52 3.2.1. Transposon Transformation Using the Epicentre® EZ-Tn5TM <KAN-2> Insertion Kit……………………………………………………………………………………….52 3.2.1.1. Sodalis glossinidius….………….……….……….……….……..….……….………52 3.2.1.2. Sodalis praecaptivus…..…….….…..….…….….…….……….…….…….….….56 3.2.2. Red Homologous Recombination of Sodalis glossinidius Using the pKD46-pKD3 System………………………………………………………………………….57 3.3. Results…….………….………….………………………………………………………………………..74 3.3.1. Transposon Transformation Using the Epicentre® EZ-Tn5TM <KAN-2> Insertion Kit……………………………………………………………………………………….74 3.3.2. Red Homologous Recombination of Sodalis glossinidius Using the pKD46-pKD3 System………………………………………………………………………….79 3.4. Discussion….……..…………..….………….…….…….…..………………………………………..84 I 4. Method Development for Sodalis glossinidius Infection of Insect Cell Lines….86 4.1. Introduction………...……….…………….…………………………………………………………..86 4.2. Methods………………..…………………………………………………………………………………88 4.2.1. Sodalis glossinidius-Development of Cell Line Infection Protocols……..88 4.3. Results………….………….…………….………………………………………………………………..91 4.3.1. Sodalis glossinidius-Development of Cell Line Infection Protocols……..91 4.4. Discussion…………….…………….…….……….………….…………………………………………99 5. Transposon-Directed Insertion Site Sequencing (TraDIS) of Sodalis glossinidius……………………………………………………………………………………………………101 5.1. Introduction….…....……….………….….…………….…..….…………….……………………101 5.2. Methods…………………………………………………………………………………………………104 5.2.1. Transposon Transformation of Sodalis glossinidius……….……….………..104 5.2.1.1. Extraction of the pRL27 Construct……………………………………………104 5.2.1.2. Transformation of Sodalis glossinidius with pRL27…………………..105 5.2.1.3. Confirmation of Transposon Insertion……………………………………..107 5.2.2. Selection Pressures for the Transposon-Directed Insertion Site Sequencing (TraDIS) Sodalis glossinidius Library………………………………110 5.2.2.1. Input Pool………………………………………………………………………………..110 5.2.2.2. Complex Medium Output Pool……….………….………….………………..112 5.2.2.3. Minimal Medium Output Pool……….……….……….………………………113 5.2.2.4. S2 Cell Line Output Pool……….…………..….…….….……………………….114 5.2.3. Library Preparation for Sequencing…….……….….….….…….……….…….….115 5.2.4. Bioinformatic Analysis……………………………………………………………………..120 5.2.4.1. Initial Quality Control……….………….………….………………………………120 5.2.4.2. Bio-TraDIS Pipeline Overview…….……………..………….….……….…….120 5.2.4.3. Bio-TraDIS Adaptation for the Sodalis glossinidius TraDIS Library……………………………………………………………………………………..122 5.2.4.4. Further Analysis…………….………….………….……….…….…….……………124 5.3. Results……….…..…………..….……………..……….………………….….………………………126 5.3.1. Bioinformatic Analysis; Bio-TraDIS Adaptation for the Sodalis glossinidius Transposon-Directed Insertion Site Sequencing (TraDIS) Library……………………………………………………………………………………………..126 5.3.2. Essential Gene Candidates for Sodalis glossinidius in vitro Growth in a Complex Medium…………………………………………………………………………….127 5.3.2.1. Essential Gene Candidates for Sodalis glossinidius Long-Term Establishment in a Complex Medium………………………………………128 5.3.2.2. Essential Gene Candidates for Sodalis glossinidius Growth in a Complex Medium versus a Minimal Medium…………………………..130 5.3.3. Essential Gene Candidates for Sodalis glossinidius in vitro Growth in a Minimal Medium versus a Complex Medium…………………………………..133 5.3.4. Overall Essential Gene Candidates for Sodalis glossinidius.…....……….135 5.3.4.1. Clusters of Orthologous Groups (COG) Analysis……………………….136 5.3.4.1.1. Metabolism……….………….…………….…….………..………………….138 5.3.4.1.1.1. Amino Acid Transport and Metabolism (COG E)………138 5.3.4.1.1.2. Carbohydrate Transport and Metabolism (COG G)….145 5.3.4.1.1.3. Inorganic Ion Transport and Metabolism (COG P)……148 5.3.4.1.1.4. Energy Production and Conversion (COG C)…………….149 II 5.3.4.1.2. Information Storage and Processing………………………………..150 5.3.4.1.3. Cellular Processes and Signalling……………………………………..151 5.3.4.2. Reciprocal Basic Local Alignment Search Tool (BLAST®) Analysis……………………………………………………………………………………151 5.3.5. Validation………………………………………………………………………………………..153 5.4. Discussion………………………………………………………………………………………………154 6. General Discussion……………………………………………………………………………………….158 References………………………………………………………………………………………………………….163 Appendix…………………………………………………………………………………………………………….180 III Acknowledgements ____________________________________________________________________ Firstly, I would like to thank my wonderful family and best friend Gill for their love and support, without which I would not have completed this journey. You all kept me going and for that, I will be forever grateful. I would also like to thank my friends Chris, Paul, Tom, Mary, Mike, Nomes, Poppy, Johnny, Stefan and Pilgrim for providing much-needed respites through the pastime of RPGs. Thank you to my supervisor Prof. Alistair Darby for his guidance and expertise throughout this project. I would also like to acknowledge the Darby Group both present and past, for their support: Fran, Dave, Sam, Joe, Gwen and Alex. By extension I would also like to thank Dr. Ian Goodhead for his Sodalis wisdom and expertise. A particular thank you to Mark, of whom started his project at the same time and has been there for me throughout. I would also like to thank my secondary supervisor Prof. Jay Hinton, of whom has always treated me like one of his own group. Within the Hinton Group I would particularly like to thank Rocio and Nico, who’s expertise proved invaluable to my project. Thank you to my tertiary supervisor Prof. Greg Hurst, and Pol and Eva within his research group, who’s symbiont and cell line insights were much appreciated. Stef and Blanca, your love and strength meant the world to me and I shall always treasure our coffee breaks. I would also like to acknowledge the University of Liverpool and the BBSRC for providing me with this opportunity. Sam, words will never do justice as a ‘thank you’. You pulled me out of the hole I found myself in and I completed this thesis because of your love and unwavering belief in me. I look forward to the rest of our lives, together. IV Abbreviations ____________________________________________________________________ Aedes albopictus larvae cells (C6/36) Adenosine triphosphate-binding cassette (ABC) Adenosine triphosphate (ATP) Analysis of Variance (ANOVA) Animal African trypanosomiasis (AAT) Antimicrobial peptides (AMPs) Bacterial artificial chromosome (BAC) Base pair (bp) Binary alignment map (BAM) Basic Local Alignment Search Tool (BLAST®) Cationic antimicrobial peptide (CAMP) Card agglutination test for trypanosomiasis (CATT) Centimetre (cm) Citric acid cycle (CAC) Clusters of Orthologous Group (COG) Coding sequence (CDS) Colony forming units (CFU) Contour-clamped homogeneous electric field (CHEF) Cytoplasmic incompatibility (CI) Deionised water (dH2O) Dimethyl sulfoxide (DMSO) Deoxyribonucleic acid (DNA) Differentially expressed (DE) Double-stranded deoxyribonucleic acid (dsDNA) Drosophila Schneider 2 cells (S2) Early log phase (ELP) Ectoperitrophic space (ES) Electron transport chain (ETC) Foetal bovine serum (FBS) V Flux Balance Analysis (FBA) Gram (g) Green fluorescent protein (GFP) Henrietta Lacks (HeLa) Human African trypanosomiasis (HAT) Hydroxymethyl pyrimidine pyrophosphate (HMP-PP) Inducible nitric oxide
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