Daniel, NeillMall, HorsburghMonshi, Manal Solayman2025-11-242025https://hdl.handle.net/20.500.14154/77140Streptococcus pneumoniae is both a commensal of the upper respiratory tract and a leading cause of pneumonia and invasive diseases. Its ability to adapt to the biochemically distinct environments of the nasopharynx and lungs is crucial for persistence and pathogenesis. This thesis investigates how S. pneumoniae metabolically adapts to these niches, with the central hypothesis that local nutrient availability and niche-specific pressures shape the bacterium’s metabolic strategies and evolutionary trajectories. To explore this, pneumococci were subjected to experimental evolution in murine models of nasopharyngeal carriage and lung infection. Twenty lineages were generated through serial in vivo passaging and subsequently characterised using carbon utilisation profiling (Biolog assays), NMR metabolomics, genome sequencing, transcriptional analyses, and in vivo infection models. Metabolomic profiling revealed clear differences in nutrient composition between the nasopharynx and lungs, particularly in the availability of carbohydrates and amino acids. Nasopharyngeal tissues were rich in host-derived glycans, while the lungs were enriched in glucose, branched-chain amino acids (BCAAs), and tripeptides. Adapted lineages displayed distinct metabolic phenotypes. Nasopharynx-adapted strains acquired enhanced capacity to utilise N-acetylglucosamine (GlcNAc), driven by mutations in regulatory genes such as nagR or promoter regions of the nanB operon, leading to increased glycosidase activity and colonisation efficiency. Lung-adapted lineages exhibited broader carbon metabolism, including increased galactose utilisation. A synonymous mutation was identified in the metabolic gene gapN in a lineage that showed long-term persistence in the nasopharynx. Experiments with gapN deletion strains suggested that redox balance plays a role in colonisation. Furthermore, phenylalanine and BCAA metabolism emerged as key adaptive traits, with several nasopharynx-adapted strains showing transcriptional upregulation of BCAA transporters (livJ, brnQ) and the global regulator codY. This work demonstrates that pneumococcal metabolic adaptation is niche-specific, often driven by regulatory rather than structural genetic changes. The findings provide mechanistic insight into host-pathogen metabolic interactions and highlight experimental evolution as a powerful tool to dissect bacterial fitness in vivo. These insights lay the foundation for future therapeutic strategies targeting metabolic pathways involved in colonisation and disease progression.210enPneumococcal metabolism • Host-pathogen interactions • Metabolomics • Bacterial adaptation.Thesis