Streptococcus pneumoniae is the most common cause of bacterial illness worldwide, causing more than 1.5 million deaths anually. S. pneumoniae commonly colonises the nasopharynx of healthy individuals asymptomatically. Despite being heavily studied a complete understanding of the mechanism(s) involved in pneumococcal pathogenesis is lacking. Antibiotic resistance is becoming a major barrier in effective treatment. Current vaccines against S. pneumoniae (PCV-13 and PPSV-23) remain ineffective against untargeted strains. A complete understanding of the pathobiology of S. pneumoniae will aid development of more effective vaccines and treatments. Our recent work reports a novel, randomly switching, N6-adenosine DNA methyltransferase (the SpnD39III system) producing six different specificities (alleles A-F). These variants produce six (epigenetically regulated) phenotypes; the first description of a phasevarion (phase-variable regulon) in a Gram-positive organism. Previously reported phasevarions regulate genes mediating immune-evasion, playing key roles in virulence. However, gene expression changes mediated by the SpnD39III system have only been studied in four of the six alleles (A-D), under in vitro conditions, and the role of phasevarion switching in colonisation, carriage, and disease has never been investigated. This project aims to characterize the phenotypes of the six SpnD39III alleles, including survival in human blood, biofilm formation, adherence to human cells, and capsule production. The impact on clinically relevant traits will also be analysed: the role of SpnD39III phase-variation in vaccine candidate expression will be investigated, as will response to antibiotics (minimum inhibitory concentrations; MICs). We will also investigate the gene/protein expression profiles of all six allelic variants of the SpnD39III system (alleles A-F), complementing and enhancing our existing data. This will provide a more robust understanding of gene regulation and pathobiology in a pathogen responsible for significant global morbidity and mortality. Findings will direct and inform future vaccine development by identifying the stably expressed antigen repertoire of S. pneumoniae.