Characterization of Novel Riboswitches Regulating Virulence Gene Expression in West African Clinical Strains of Streptococcus pneumoniae: Structural Biology and Translational Inhibition

Riboswitches are cis-regulatory RNA elements in bacterial 5-prime untranslated regions that control gene expression in response to specific ligands, serving as promising antibiotic targets given their absence in eukaryotes. Streptococcus pneumoniae, a leading cause of pneumonia and meningitis in Nigerian children, harbors riboswitches whose structural diversity in West African clinical strains is unexplored. This dissertation characterizes novel riboswitches regulating virulence gene expression in West African clinical S. pneumoniae strains isolated from pediatric patients in Ekiti, Ogun, and Oyo States, Southwest Nigeria. Clinical S. pneumoniae isolates from blood and CSF cultures were whole-genome sequenced using Illumina MiSeq. Computational riboswitch prediction was performed using Rfam-based covariance model searches and RNAfold structure prediction. Six candidate novel riboswitch aptamer structures were identified upstream of virulence-associated genes (pneumolysin-ply, neuraminidase-nanA, capsular polysaccharide biosynthesis cpsB). In vitro transcription and SHAPE-MaP chemical probing were used to experimentally determine riboswitch secondary structure with single-nucleotide resolution. Ligand-binding activity was characterized by isothermal titration calorimetry (ITC) for the two highest-confidence candidates. Small molecule riboswitch inhibitor screening was performed against 1,200 FDA-approved compounds using a thermal shift assay. Two functional riboswitches were validated: a novel cobalamin-responsive riboswitch upstream of ply and an S-adenosylmethionine-responsive element upstream of nanA. High-resolution structures were obtained by X-ray crystallography at 2.1 angstrom resolution. Three FDA-approved compounds showed binding affinities below 5 micromolar to the ply riboswitch aptamer, with downstream reduction in ply expression confirmed by RT-qPCR. These findings identify novel RNA-based drug targets in West African S. pneumoniae with translational inhibition potential. Keywords: riboswitch, Streptococcus pneumoniae, virulence gene regulation, structural biology, Southwest Nigeria.