Fig. 4.

Entry through clathrin independent pathways promotes improved intracellular persistence of SPN. (A) Intracellular survival efficiency of WT SPN in hBMECs following pre-treatment with combination of endocytosis inhibitors (Dynasore, MβCD + EIPA and CPZ + EIPA). Exponentially grown SPN were allowed to infect confluent hBMEC monolayers at MOI 10 and after killing extracellular SPN with penicillin (10 µg/ml) and gentamycin (400 µg/ml), survival ability of internalized SPN at indicated time points were calculated by enumerating bacterial numbers following plating cellular lysates on BHI plates. Survival efficiency were calculated as percent survival at indicated time points relative to 0 h. Data are presented as mean ± SD of triplicate experiments. Statistical analysis was performed using two-way ANOVA (Bonferroni test). ns, non-significant; *p < 0.05; **p < 0.01, ***p < 0.001. (B) Confocal micrographs showing association of SPN (green) with lysosomal marker cathepsin B (red) at 6 h post infection. hBMECs were infected with SPN and stained with anti-Cathepsin B Ab. Multiple images (n ≥ 50) were captured using laser scanning confocal microscope and representative images are shown. White arrow designates SPN present inside lysosomes, while blue arrow depicts non-lysosome associated SPN. Scale bar, 5 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)