Fig. 2.

Pneumococcus adopts dynamin independent pathway for entry into the brain endothelium. Invasion (A-C) and adherence (D-F) of SPN to hBMECs as measured by gentamicin protection assays following treatment with various concentrations of dynamin inhibitors Dynasore (A, D) and Dyngo-4a (B, E) or dynamin independent pathway inhibitor EIPA (C, F). Exponentially grown SPN were allowed to infect confluent hBMEC monolayers at MOI 10 and adherence and invasion assays were performed as described in methods. The levels of invasion or adherence were normalized to vehicle treated cells. (G) Verification of pneumococcal cell surface protein shaving following trypsinization by SDS-PAGE. Lane 1: Mol. wt. marker; Lane 2: SPN lysate; Lane 3: lysate of trypsin treated SPN; Lane 4: supernatant of trypsin treated SPN. (H) Role of cell surface proteins in pneumococcal invasion of brain endothelium through dynamin independent pathway. Chloramphenicol (0.85 µg/ml, for blocking protein synthesis) treated SPN was trypsinized (1 µg/ml) and allowed to invade hBMECs through dynamin independent pathway following inhibition of dynamin dependent endocytosis with Dynasore (100 μM). The levels of invasion were normalized to vehicle treated cells. In all cases, results are represented as mean ± SD of triplicate experiments and representative graphs are shown. Statistical tests were performed using One-way ANOVA followed by Tukey's multiple comparison test. ns, non-significant; *p < 0.05; ***p < 0.001.