Impact ofStreptococcus pneumoniaebiosynthesis gene mutations on epithelial microinvasion and cellular responses

ABSTRACT The influence of Streptococcus pneumoniae metabolism on nasal epithelial interactions is under-explored. We have assessed S. pneumoniae mutations affecting glutamate 5 kinase ( proABC ) and formate tetrahydrofolate reductase ( fhs ). A Pia iron transporter ( piaA ) mutation was added to ensure attenuation in Experimental Human Pneumococcal Challenge (EHPC). Epithelial microinvasion by Δ proABC/piaA and Δ fhs/piaA strains was enhanced compared to wild-type (WT) in EHPC and Detroit 562 cell infection. In primary epithelium, acetylated tubulin and β catenin expression was increased following Δ proABC exposure and uteroglobin expression was increased with Δ fhs exposure. In Detroit 562 cells, WT and Δ proABC infection induced an inflammatory epithelial transcriptomic response. The Δ fhs/piaA strain induced primarily cellular stress/ repair gene responses. Differential caspase 3/7/8 activity appeared linked to pneumococcal pneumolysin activity and hydrogen peroxide secretion. Our findings highlight the broad-ranging effects of single gene mutations in pneumococcal metabolism which underlies the complexity of the differences between seemingly closely related pneumococci. GRAPHICAL ABSTRACT Bacterial transcriptomics revealed profound effects of biosynthesis gene mutations on bacterial gene expression under serum stress, with upregulation of bacterial virulence factors such as pneumolysin and hydrogen peroxide regulator genes (e.g. spxB) . Epithelial microinvasion by the isogenic mutants was increased compared to wild type strain, but bacterial load was not sufficient to account for the differences observed in epithelial gene expression. Deletion of the fhs gene induced a more intrinsic cellular stress and repair response, compared to the wild-type strain or deletion of the proABC operons which induced a more typical pro-inflammatory response. Differential effects were also seen in cellular re-modelling and caspase activity. We postulate that this link between bacterial metabolism and virulence under conditions of stress will ultimately influence the outcome of colonisation in terms of transmission potential, transition to disease and generation of protective immunity.