PPaatthhooggeenneessiiss ooff SSttrreeppttooccooccccuuss ppnneeuummoonniiaaee Lauren Joy McAllister, BSc (Mol. Biol.), BSc (Hons) A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy from the University of Adelaide May, 2010 Research Centre for Infectious Diseases, School of Molecular & Biomedical Sciences, University of Adelaide, Adelaide, S.A., Australia This thesis is dedicated to my mother, and sisters Janine and Suzanne. Page | i Table of Contents Abstract ·············································································· x Declaration ········································································ xiii Acknowledgements ···························································· xiv List of Abbreviations ·························································· xvii Chapter 1: General Introduction 1.1 Historical Background ··························································································· 1 1.2 Pneumococcal Disease ························································································· 3 1.3 Pneumococcal Virulence ······················································································· 6 1.3.1 The pneumococcal capsule ····················································································· 8 1.3.2 Serotype, sequence type and genome ······································································· 9 1.3.3 Virulence proteins ·································································································· 16 1.3.3.1 Pneumococcal surface protein A (PspA) ····························································· 16 1.3.3.2 Pneumococcal surface protein C (PspC) ···························································· 16 1.3.3.3 Neuraminidase A (NanA) ················································································· 17 1.3.3.4 Pneumolysin (Ply) ·························································································· 18 1.3.3.4 Autolysin (LytA) ····························································································· 18 1.3.4 Phase variation ····································································································· 19 Table of Contents Page | ii 1.3.5 Physiological states ······························································································· 21 1.4 Otitis Media ········································································································· 24 1.4.1 OM pathogenesis ·································································································· 25 1.4.3 OM in Australian Aboriginal children ·········································································· 26 1.4.4 Control of OM: current and future strategies ······························································· 28 1.4.5 OM and the pneumococcus ····················································································· 31 1.4.5.1 Serotype and genotype ··················································································· 31 1.4.5.2 OM-associated proteins ·················································································· 32 1.4.5.3 Phase variation in OM ····················································································· 33 1.4.5.4 Biofilms in OM ······························································································· 34 1.5 Aims of the Project ······························································································· 35 Chapter 2: Materials and Methods 2.1 General Chemicals and Reagents ······················································································· 37 2.1.1 Antibiotics ······················································································································· 37 2.2 Bacterial Strains and Growth ······························································································ 38 2.2.1 S.pneumoniae Strains ······································································································· 38 2.2.2 Pneumococcal Growth and Storage ····················································································· 41 2.2.3 Optochin sensitvity ··········································································································· 41 2.2.4 Quellung Reaction ············································································································ 42 2.2.5 Determination of colony opacity phenotype············································································ 42 2.2.6 Uronic acid assay ············································································································· 42 2.2.7 Determination of growth curves ··························································································· 43 2.2.8 Purple broth assay ··········································································································· 44 Table of Contents Page | iii 2.2.9 Biofilm assay ··················································································································· 44 2.2.10 Passaging of pneumococcal strains ··················································································· 45 2.2.11 E.coli strains, growth and storage ······················································································ 45 2.3 Oligonucleotide Primers ···································································································· 46 2.3.2 Oligonucleotide Adaptors ··································································································· 53 2.4 Protein Analysis ··············································································································· 54 2.4.1 Preparation of whole cell lysates ························································································· 54 2.4.2 SDS-PAGE ····················································································································· 54 2.4.3 Western Blot Analysis ······································································································· 54 2.4.4 Haemolysis Assay ············································································································ 55 2.5 DNA Isolation and Manipulation ·························································································· 56 2.5.1 Agarose Gel Electrophoresis ······························································································ 56 2.5.2 Pneumococcal chromosomal DNA isolation ··········································································· 57 2.5.3 Pneumococcal chromosomal DNA isolation using phenol ························································· 57 2.5.4 Restriction endonuclease digestion of DNA ··········································································· 58 2.5.5 Polymerase chain reaction (PCR) ························································································ 58 2.5.6 Purification of PCR products and Restriction Digests ······························································· 59 2.5.7 Cloning of PCR product ····································································································· 59 2.5.8 Plasmid Miniprep ············································································································· 60 2.5.9 DNA sequencing ·············································································································· 60 2.5.10 Next generation sequencing ····························································································· 61 2.5.11 Bioinformatics ················································································································ 61 2.6 Multi Locus Sequence Typing ···························································································· 62 2.7 DNA Microarray ················································································································ 62 2.8 PCR-Based Subtraction Hybridisation ················································································· 63 Table of Contents Page | iv 2.9 Southern Hybridisation ······································································································ 64 2.9.1 Prepartion of membrane ···································································································· 64 2.9.2 Hybridisation ··················································································································· 64 2.9.3 Generation of probes ········································································································ 65 2.9.4 Development of the Membrane ··························································································· 65 2.10 RNA Isolation and Manipulation ························································································ 65 2.10.1 Hot-Phenol RNA Extraction ······························································································ 65 2.10.2 Reverse Transcription-PCR (RT-PCR) ················································································ 67 2.10.3 Real-time RT-PCR ··········································································································