Low Ply expression promotes pneumococcal trafficking across BBB.

<p>Balb/c mice were infected via i.v. route with 10⁶ CFU of WT, WT:Ply-High or WT:Ply-Low SPN strain and at 14 h p.i. animals were sacrificed. PBS injected mice served as control. <b>A.</b> Confocal micrographs depicting heterogeneous Ply expression (red) within WT SPN population (blue) in the blood of infected mice. Blood smears were stained with Anti-Ply antibody and DAPI. Scale bar, 5 μm. <b>B.</b> Ratio of transcript levels of Ply in brain to blood of mice infected with WT SPN. Transcript levels were normalized to 16S rRNA and expressed as fold change compared to Ply transcript levels of WT SPN grown <i>in vitro</i> in THY medium. <b>C.</b> Kaplan-Meier survival curve of mice (n = 5 per group) following i.v. injection with different SPN strains. Experiments were repeated twice and one representative graph is shown. Statistical analysis was performed using Log-rank test. <b>D.</b> Net growth of SPN strains in the blood of infected mice (n = 5 per group) at indicated time points post infection. Experiments were repeated twice and one representative graph is shown. Statistical analysis was performed using two-way ANOVA (Bonferroni test); ns, non-significant. <b>E.</b> Quantification of bacterial counts (CFU) in various tissue homogenates and blood of mice infected with different Ply expressing SPN strains. Each dot represents one mouse; black bars show average values. n = 12 per group (2 experiments with n = 6 per group). Statistical analysis for each tissue was individually performed using non-parametric test (Mann-Whitney test). <i>p</i> values are mentioned in the graph. <b>F—H.</b> Ratio of bacterial CFU in brain to blood <b>(F)</b>, lungs to blood <b>(G)</b> and spleen to blood <b>(H)</b> of individual infected mice. Each dot represents one mouse; black bars show average values. n = 12 per group (2 experiments with n = 6 per group). Statistical analysis was performed using non-parametric test (Mann-Whitney test). <i>p</i> values are mentioned in the graph. <b>I.</b> Transcript abundance of pro-inflammatory cytokines in total RNA isolated from brains of mice that were injected with PBS or infected with different SPN strains. Transcript levels were normalized to 18S rRNA and expressed as fold change compared to control mice. Statistical analysis was performed using two-way ANOVA (Bonferroni test); ns, nonsignificant. <b>J.</b> Histopathology of H & E stained representative brain tissue samples of control (PBS injected) or mice infected with different strains of SPN. Scale bar, 100 μm.