Impact of Streptococcus pneumoniae biosynthesis gene mutations on epithelial microinvasion and cellular responses

Epithelial cells are important gatekeepers during bacterial colonisation of the nasopharynx by Streptococcus pneumoniae. The bacterial and host factors influencing the outcomes of this interaction are poorly understood but there is increasing recognition that bacterial metabolism is an important determinant of virulence. Pneumococcal colonisation is associated with microcolony formation and epithelial microinvasion without disease in healthy individuals. We have assessed the impact of mutations in two S. pneumoniae biosynthesis genes, glutamate 5 kinase ( proABC ) and formate tetrahydrofolate reductase ( fhs ) on epithelial cell microinvasion and subsequent epithelial-innate immunity, using an Experimental Human Pneumococcal Challenge (EHPC) model, primary NHBE-A healthy human bronchial epithelial cells and Detroit 562 nasopharyngeal cells. Bacterial transcriptomic analysis revealed profound effects to stress response following deletion of proABC or fhs genes. Mutant strains retained the ability to colonise in the EHPC model. By day 6 post-inoculation, a higher proportion of mutant strains were found inside epithelial cells compared to WT. In primary epithelial cells, microscopy analyses revealed epithelial surface differences between strains, with enhancement of acetylated tubulin (a marker of Ciliated cells) and β catenin expression following Δ proABC/piaA exposure and enhancement of uteroglobin (a marker of Clara cells) expression following Δ fhs/piaA exposure. In Detroit 562 cells, pneumococcal adhesion was reduced ten-fold for both the Δ proABC/piaA and Δ fhs/piaA strains compared to WT. Microinvasion was four times higher for the mutants compared to WT. In Detroit 562 cells, the WT and Δ proABC/piaA strains induced a similar epithelial transcriptomic response, dominated by inflammatory response genes, distinct from the response to Δ fhs/piaA strain, which primarily induced upregulation of cellular stress and repair genes. Insights into differential activation of caspase pathways following infection with the different strains shed light on pneumococcal triggering of epithelial-derived innate immunity. These differential epithelial cell responses may in part be due to differences in effects on pneumococcal metabolic pathways, activity of pneumolysin and hydrogen peroxide production, between the strains. Despite almost identical colonisation profiles to WT, just a single bacterial gene mutation in metabolic genes revealed widespread and differing effects on pneumococcal-epithelial interactions and epithelial cell responses. Overall, our findings suggest that it is bacterial metabolism and virulence, but not pneumococcal load thresholds, that determine the epithelial host response. We propose this new paradigm has important implications for pneumococcal niche adaptation, transmission and transition towards disease. GRAPHICAL ABSTRACT Bacterial transcriptomics reveals profound changes in bacterial gene expression under stress, for which biosynthesis genes play a major role. During serum-induced stress and deletion of proABC or fhs genes, bacterial virulence factors such as ply and hydrogen peroxide regulator genes such as spxB were upregulated. Using a range of different cell culture models, all pneumococcal strains were able colonise and micro-invade epithelial cells. Bacterial load was not sufficient to account for the differences observed in epithelial gene expression, following infection with the pneumococcal strains. Epithelial cell responses were instead dependent upon bacterial adaptation profiles during nasopharyngeal colonisation. For example, deletion of the fhs gene induced a more intrinsic and epithelial repair response, enriching genes such as MAPK3/7, compared to 6B WT and deletion of the proABC gene which induced a more typical pro-inflammatory immune response, enriching genes such as RELA. The combination of host-pathogen interactions strengthens the link between bacterial metabolism and virulence which will ultimately influence the outcome of colonisation, transmission potential, transition to disease and generation of protective immunity. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes