Genomic Diversity in Naturally Transformable Streptococcus pneumoniae Dr Donald James Inverarity BSc MBChB MRCP MSc DTMH DLSHTM FRCPath A Thesis Submitted in Fulfilment of the Requirements for the Degree of PhD Division of Infection and Immunity Faculty of Biomedical and Life Sciences University of Glasgow February 2009 © Donald James Inverarity 2 Abstract Infections due to Streptococcus pneumoniae (the pneumococcus) remain a substantial source of morbidity and mortality in both developing and developed countries despite a century of research and the development of effective therapeutic interventions (such as antibiotic therapy and vaccination). The ability of the pneumococcus to evade multiple classes of antibiotic through several genetically determined resistance mechanisms and its evasion of capsular polysaccharide based vaccines through serotype replacement and capsular switching, all reflect the extensive diversity and plasticity of the genome of this naturally transformable organism which can readily alter its genome in response to its environment and the pressures placed upon it in order to survive. The purpose of this thesis is to investigate this diversity from a genome sequence perspective and to relate these observations to pneumococcal molecular epidemiology in a region of high biodiversity, the pathogenesis of certain disease manifestations and assess for a possible bacterial genetic basis for the pneumococcal phenotypes of, “carriage” and, “invasion.” In order to do this, microarray comparative genomic hybridization (CGH) has been utilized to compare DNA from a variety of pneumococcal isolates chosen from 10 diverse serotypes and Multilocus Sequence Types and from clinically relevant serotypes and sequence types (particularly serotypes 3, 4 and 14 and sequence types ST9, ST246 and ST180)) against a reference, sequenced pneumococcal genome from an extensively investigated serotype 4 isolate – TIGR4. Microarray comparison of the transcriptional profiles of several isolates has also been undertaken to compare gene expression from isolates of serotype 1 (ST227 and ST306) and serotype 3 (ST180) related to particular disease states and exposure of a multi-resistant pneumococcus to an antimicrobial (clarithromycin) commonly used to treat pneumococcal pneumonia. 3 Table of Contents Abstract ..................................................................................................................................2 List of Tables..........................................................................................................................7 List of Figures ........................................................................................................................9 Acknowledgements..............................................................................................................12 Author’s Declaration............................................................................................................14 Abbreviations .......................................................................................................................15 1 Introduction..................................................................................................................18 1.1 Taxonomy of Streptococcus pneumoniae ............................................................18 1.2 Phenotypic identification and characteristics of Streptococcus pneumoniae ......19 1.3 Genetic typing of Streptococcus pneumoniae......................................................21 1.4 Pneumococcal Carriage........................................................................................28 1.5 Manifestations of Pneumococcal Infection..........................................................31 1.6 Studies of the Genomic Diversity of Streptococcus pneumoniae........................35 2 Materials and Methods.................................................................................................63 2.1 General Procedures ..............................................................................................63 2.2 Microarray Protocols............................................................................................67 2.3 Gene Sequencing and Multi Locus Sequence Typing .........................................72 2.3.1 Gene Sequencing..............................................................................................72 2.3.2 Multi Locus Sequence Typing .........................................................................73 2.4 Analysis of Genome Sequences Using the Artemis Comparison Tool................73 2.5 Quantitative Real Time Polymerase Chain Reaction...........................................74 2.5.1 cDNA Synthesis...............................................................................................74 2.5.2 Quantitative Real Time Polymerase Chain Reaction Protocol ........................74 3 Microarray and Genome Sequencing Approaches to the Study of Pneumococcal Genomic Diversity ...............................................................................................................76 3.1 What is a Microarray?..........................................................................................76 3.2 Applications of Microarrays ................................................................................77 3.3 Microarray Design ...............................................................................................79 3.4 Microarray Analysis Methods..............................................................................80 3.5 Microarray Validation Experiments.....................................................................82 3.5.1 Validation of DNA CGH .................................................................................82 3.6 Comparisons of Microarray DNA CGH results with Sequenced Pneumococcal Genomes...........................................................................................................................89 3.6.1 Choice of Diverse Sequenced Strains ..............................................................89 3.6.2 Comparison of Microarray DNA CGH results with Genome Sequence Data.89 3.6.3 Genomic Diversity Identified by Genome Sequence Data Alone ...................94 3.6.4 Using Pneumococcal Genome Sequences to Identify Gene Insertions ...........97 3.7 Discussion ..........................................................................................................100 3.7.1 Advantages and Disadvantages of a Genome Sequencing Approach to mapping Genomic Diversity compared to microarray based CGH investigations....100 3.7.2 The Distributed Genome Hypothesis and a Pneumococcal Supragenome ....102 3.7.3 Microarray Limitations ..................................................................................102 4 Genomic Diversity Observed in Phenotypically Diverse Pneumococcal Isolates.....104 4.1 Choice of Diverse Pneumococcal Isolates .........................................................104 4.2 Microarray Results.............................................................................................106 4.3 Discussion ..........................................................................................................106 4.3.1 Regions of Diversity in the TIGR4 and R6 Genomes....................................106 4.3.2 Diversity at Particular Genetic Loci...............................................................111 4.4 Conclusions........................................................................................................115 5 Genomic Diversity in a Multilocus Sequence Type Associated with Invasive Pneumococcal Disease (IPD).............................................................................................116 4 5.1 Serotype 14 and ST9: associations with IPD .....................................................116 5.2 Choice of Isolates...............................................................................................116 5.3 Microarray Results.............................................................................................117 5.4 Discussion ..........................................................................................................117 5.4.1 Overall diversity in ST9.................................................................................117 5.4.2 Regions of Diversity in the TIGR4 and R6 genomes ....................................120 5.4.3 Diversity at Particular Genetic Loci...............................................................120 6 Genomic Diversity within a Multilocus Sequence Type Accounting for Invasive Pneumococcal Disease and Carriage .................................................................................123 6.1 A Review of the Associations of Serotype 3 and ST180 with Pneumococcal Carriage or Invasive Disease..........................................................................................123 6.2 Serotype 3 ST180 Isolates Analysed by Microarray CGH ................................124 6.3 DNA CGH Comparison of Carriage and Invasive Isolates of ST180 ...............125 6.4 RNA Expression Differences in Carriage and Invasive Isolates of ST180 .......127 6.4.1 Microarray results ..........................................................................................127 6.4.2 Quantitative Real Time PCR results ..............................................................129 6.5 Discussion ..........................................................................................................130