SPD_1629 is a uracil transporter and is crucial for pneumococcal fitness and pathogenesis in vivo.

Streptococcus pneumoniae (pneumococcus), a major human pathogen, causes pneumonia, bacteraemia and meningitis. Unlike free-living bacteria such as Escherichia coli, pathogenic bacteria like S. pneumoniae possess incomplete metabolic pathways and depend on host-derived nutrients. While nucleotide metabolism is essential for bacterial proliferation, how S. pneumoniae takes up uracil is still not well understood. Identifying uracil transporters is important, as they may serve as targets for novel antimicrobial strategies. In this study, we functionally characterized a uracil transporter using the toxic uracil analog 5-fluorouracil (5-FU). In vitro assays showed dose-dependent inhibition of bacterial growth by 5-FU, which was reversed by excess uracil, suggesting the presence of a specific uracil transporter. Bioinformatic analysis identified SPD_0267, SPD_1141 and SPD_1629 as candidates. A mutant lacking SPD_1629 (D39Δspd_1629) showed partial resistance to 5-FU, while deletion of SPD_0267 or SPD_1141 had no effect. The growth of the triple mutant D39Δspd_0267Δspd_1141Δspd_1629 mirrored that of D39Δspd_1629. Unlike in rich medium, D39Δspd_1629 mutant failed to grow in uracil-sufficient chemically defined medium. Radiolabeled [³H]-uracil uptake assays validated the transporter's specificity for uracil. In vivo, D39Δspd_1629 displayed significantly reduced bacterial loads in the lungs, blood and spleen of infected mice. Competitive index assays further revealed a severe fitness defect relative to the wildtype. spd_1629 deletion also reduced capsular polysaccharide levels, correlating with attenuated virulence in a mouse model of pneumonia and sepsis. These findings establish SPD_1629 as the principal uracil transporter in S. pneumoniae, and highlight its critical role in virulence and in vivo fitness.