Contribution of Glucose to Increased Respiratory Bacterial Burden in Hyperglycaemia Simren Kaur Gill Department of Medicine, Imperial College London Thesis submitted for the degree of Doctor of Philosophy 1 Abstract It has recently been proposed that uncontrolled hyperglycaemia in people with diabetes increases lung glucose, so providing a richer growth medium facilitating bacterial infection. Where diabetes is controlled, there is a reduced associated risk of bacterial infection. The aim of this thesis was to determine the effect of glucose on bacterial growth in vivo and directly link bacterial glucose metabolism with increased respiratory tract bacterial load in hyperglycaemia. P. aeruginosa, a Gram-negative opportunistic pathogen, which is a major cause of respiratory infections, was used as chronic P. aeruginosa infections are most commonly associated with people with cystic fibrosis (CF) and chronic obstructive pulmonary diseases (COPD), but importantly P. aeruginosa is increasingly diagnosed in diabetic patients with pneumonia/lung infection. To test the hypothesis, P. aeruginosa mutants were generated by deleting oprB, gltK, gtrS and glk genes and these mutant strains were used in in vitro and in vivo infection models developed during this thesis. The mutants had drastically reduced growth in minimal medium containing glucose as the sole carbon source, whereas they were unaltered when grown in rich medium. In order to explore the effect of elevated glucose with minimal effects on the immune response, streptozocin was used to induce diabetes, instead of genetically obese mice, which have a complex phenotype and impaired immune responses. Streptozocin induced hyperglycaemia also led to increased bacterial load in the airways when mice were infected with wild type PAO1 but not with the glucose uptake and metabolism mutants. To further support this hypothesis when metformin was used to lower glucose levels it resulted in a reduced bacterial burden. 2 Acknowledgements Firstly I would like to thank Dr John Tregoning, without whom this project would not have been possible, his guidance throughout, both scientifically and as a mentor has been invaluable and I am grateful for having such an inspiring and motivational supervisor. John was instrumental in encouraging me to continue in scientific research and I hope to have the pleasure of collaborating with him in the future. I would also like to thank Professor Alain Filloux and members of his lab, particularly Dr Abderrahman Hachani and Kailyn Hui whose expertise in mutant generation was critical for the success of this project. Professor Deborah Baines and Dr James Garnett from St George’s University London, who assisted with the development of the in vitro infection models and Dr Kai Hilpert for providing the peptides as well as Akshay Sabnis who helped with the development of the TraDIS library. Special thanks are also extended to the Shattock Group, it’s been fun. This project was funded by the Wellcome Trust and the Institutional Strategic Support Fund. 3 Declaration of Originality The work presented in this thesis is my own and where appropriate all references are cited. Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. Word Count: 33 108 (excluding bibliography) 4 Table of Contents Contents Abstract .................................................................................................................................... 2 Acknowledgements ................................................................................................................. 3 List of Figures and Tables ....................................................................................................... 9 Abbreviations ......................................................................................................................... 10 1. Introduction ................................................................................................................... 12 1.1. Respiratory Bacterial Infections.............................................................................. 12 1.2. Signs and symptoms................................................................................................ 13 1.3. Clinical Infections with P. aeruginosa .................................................................... 14 1.4. Antimicrobial Resistance ........................................................................................ 14 1.5. Pseudomonas ........................................................................................................... 18 1.5.1. Pseudomonas aeruginosa ................................................................................. 18 1.5.2. Pseudomonas Genome ..................................................................................... 19 1.5.3. P. aeruginosa Virulence Mechanisms During Acute infection ........................ 19 1.5.4. Chronic Infection and Biofilm Formation ....................................................... 24 1.6. Acute and Chronic Infection ................................................................................... 26 1.7. Carbon Utilisation by P. aeruginosa ....................................................................... 27 1.8. Risk Factors for infection ........................................................................................ 31 1.8.1. Cystic Fibrosis (CF) ......................................................................................... 31 1.8.2. Chronic Obstructive Pulmonary Disorder (COPD) ......................................... 32 1.8.3. Obesity ............................................................................................................. 33 1.8.4. Diabetes ........................................................................................................... 34 1.8.5. Hyperglycaemia ............................................................................................... 35 1.9. Therapies against P. aeruginosa .............................................................................. 36 1.9.1. Antimicrobial resistance in P. aeruginosa ....................................................... 37 1.9.2. Potential Alternative Therapies ....................................................................... 38 1.10. Host Defence ....................................................................................................... 39 1.10.1. Airway Surface Liquid ................................................................................. 39 1.10.2. Physical Defence mediated by Epithelial Cells ........................................... 41 1.10.3. Pattern Recognition of P. aeruginosa ........................................................... 41 1.10.4. Cytokine Release by Airway Epithelial Cells .............................................. 43 1.10.5. Macrophages ................................................................................................ 44 1.10.6. Neutrophils ................................................................................................... 46 1.10.7. Eosinophils ................................................................................................... 46 1.11. Adaptive Immune Response ................................................................................ 47 5 1.12. Glucose homeostasis and the role of glucose in immune responses ................... 47 1.12.1. Glucose and Bacterial Infection Previous studies ........................................ 49 1.13. Hypothesis and Aims ........................................................................................... 53 1.13.1. Hypothesis .................................................................................................... 53 1.13.2. Aims................................................................................................................. 53 2. Materials and Methods .................................................................................................. 54 2.1. Reagents List ........................................................................................................... 54 2.2. Microbiology Techniques ....................................................................................... 57 2.2.1. Bacterial Strains ............................................................................................... 57 2.2.2. Glycerol Stocks ................................................................................................ 58 2.2.3. Cultures ............................................................................................................ 58 2.2.4. Colony Forming Units/ml Calculations ........................................................... 58 2.2.5. Growth and Dose Preparation .......................................................................... 59 2.2.6. Characterisation of Bacterial Strains in Different Growth Media ................... 59 2.2.7. High Throughput Growth of Bacteria ............................................................. 60 2.2.8. Mutant generation ...........................................................................................