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Interaction of Bacteroides fragilis with Host Proteins and Effects of Nitrogen Limitation on the B. fragilis Transcriptome Aparna Shankar Thesis presented for the degree of Doctor of Philosophy Institute of Cell Biology The University of Edinburgh November 2015 Declaration The author performed all of the investigations and procedures presented in this thesis, unless otherwise stated. APARNA SHANKAR i Acknowledgements I would like to take this opportunity to express my sincere gratitude to each and everyone who made this four year journey worthwhile and unforgettable. To begin with, I thank my supervisor Dr. Garry Blakely for giving me an opportunity to pursue doctoral research in his lab. It was definitely his constant encouragement, valuable suggestions and enthusiasm for science that kept me going. I would also like to thank my second supervisor, Dr. Maurice Gallagher and our collaborator, Prof. Sheila Patrick for their timely inputs that led me in the right direction in my project. I extend my thanks to the Edinburgh Genomics team who supported me with the sequencing work and Ms. Persistent Bioinformatics for assisting me in the analysis. In particular, I thank Dr. Anamika and Srikant for patiently answering all my queries regarding the in silico analysis, which helped me in the project completion. I also thank the members of the Darwin media kitchen for their organised work which helped me plan my daily laboratory experiments without hindrance. A big thank you to all my past lab members and the lovely floor mates of the old Darwin building for the informative talks and discussions on science. A special hug goes out to Keerthi, Pradeep, Daksh, Sanju and Sharanya for spreading goodwill and cheer which helped me de-stress after a bad 'science' day at work! I would also like to thank all my dear friends back home who were just a call away whenever I needed them. I am extremely indebted to my family, especially my parents, mother-in-law and father-in-law for believing in me and being there by my side. I owe it to my mother for instilling a passion for science and research in me. I am short of words to thank my ever loving and caring husband, Raman, for his round-the-clock support, technical help and above all, for patiently putting up with my 'thesis-induced' panic! ii Table of Contents Chapter 1. Introduction: The Human Microbiome ................................................ 5 1.1. Commensalism in the Human Intestine ............................................................. 6 1.2. Bacteroides fragilis ......................................................................................... 10 1.2.1. Polysaccharide synthesis........................................................................... 11 1.2.2. Polysaccharide A ...................................................................................... 13 1.2.3. Outer membrane vesicles .......................................................................... 15 1.2.4. Pathogenic factors ..................................................................................... 19 Chapter 2. Materials and Methods ......................................................................... 23 2.1. Strains .............................................................................................................. 23 2.2. Bacterial culture conditions ............................................................................. 23 2.3. Oligonucleotides .............................................................................................. 24 2.4. Bacterial techniques ........................................................................................ 28 2.4.1. B. fragilis growth curves ........................................................................... 28 2.4.2. Preparation of chemically competent cells ............................................... 28 2.4.3. Transformation: Heat-shock method ........................................................ 29 2.4.4. Transformation: Electroporation method.................................................. 29 2.4.5. Conjugation of E. coli S17-1 λpir with B. fragilis NCTC 9343 .............. 30 2.4.6. Screening for deletion genotypes .............................................................. 30 2.5. DNA techniques .............................................................................................. 30 2.5.1. Genomic DNA extraction ......................................................................... 30 2.5.2. Plasmid DNA extraction ........................................................................... 30 2.5.3. Polymerase chain reactions ....................................................................... 31 2.5.4. Agarose gel electrophoresis ...................................................................... 31 2.5.5. DNA purification ...................................................................................... 31 2.5.6. DNA quantification................................................................................... 32 2.5.7. Restriction digestion ................................................................................. 32 2.5.8. Cloning...................................................................................................... 32 iii 2.5.9. Single colony assay ................................................................................... 32 2.5.10. Sanger-based plasmid sequencing .......................................................... 33 2.6. RNA sequencing .............................................................................................. 33 2.6.1. RNA extraction ......................................................................................... 33 2.6.2. Library preparation ................................................................................... 34 2.7. Protein techniques............................................................................................ 35 2.7.1. Preparation of whole cell protein extracts ................................................ 35 2.7.2. Preparation of concentrated culture supernatants ..................................... 35 2.7.3. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) ................................................................................................................. 35 2.7.4. Staining/destaining of gels ........................................................................ 36 2.7.5. Zymography .............................................................................................. 36 2.7.6. Preparation of samples for immunoblot analysis ...................................... 37 2.7.7. Transfer of proteins by wet electroblotting ............................................... 38 2.7.8. Development of western blots and far-western blots ................................ 38 2.7.9. Stripping antibodies from immunoblots ................................................... 39 2.7.10. Protein expression ................................................................................... 39 2.7.11. Protein purification by nickel affinity chromatography .......................... 41 2.7.12. Protein estimation by bradford assay ...................................................... 42 2.7.13. Immunofluorescence microscopy (IFM) ................................................ 43 2.8. Bioinformatics .............................................................................................. 44 2.8.1. Quality control of RNA extracts ............................................................... 44 2.8.2. RNA sequencing data, assembly and annotation ...................................... 44 Chapter 3. Binding of Host Proteins by Bacteroides fragilis ................................ 47 3.1. Introduction ..................................................................................................... 47 3.1.1. Structure of the extracellular matrix (ECM) ............................................. 48 3.1.2. Microbial surface components recognised by adhesive matrix molecules (MSCRAMMs) in bacteria ................................................................................. 55 3.1.3. MSCRAMMs expressed in intestinal microbiota ..................................... 61 3.1.4. Aims .......................................................................................................... 67 iv 3.2. Choice of bacterial strains ............................................................................... 68 3.3. Generation of markerless deletions in Bacteroides fragilis NCTC 9343 genome ..................................................................................................................