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. 2019 Jul 16;10:3060. doi: 10.1038/s41467-019-11005-2

Fig. 3.

Fig. 3

Pneumococcal infection in vitro is associated with adhesion, micro-colony formation and micro-invasion. Pneumococcal a association and b internalisation: Detroit 562 cell monolayers were stimulated with pneumococci for 1 or 3 h and the quantity of associated bacteria was determined by culture (CFU); n = 6. ****P = < 0.0001, ***P = 0.0001. a 1 h (dashed lines, ANOVA); 3 h (solid lines, Kruskall–Wallis); b (Kruskall–Wallis). c Pneumococcal transmigration: Cells on transwell inserts were stimulated with pneumococci for 3 h and bacterial density in the basal chamber was determined (**P = 0.0092, Kruskall–Wallis). N = 5. a–c Error bars represent s.e.m. d Representative pneumococcal-density (green) images of cells (blue nuclei). Scale bar = 20 µm. n = 5. e–h Representative images of pneumococcal localization (green) from cells (red, JAM-A), illustrating: e differences in adherence by 6B and TIGR4; f micro-colony and chain formation (green) dPly-TIGR4 (green) and WGA (red); g internalised bacteria (top 6B; bottom TIGR4) co-localised with JAM-A with associated intracellular vesicle-like bodies (yellow), or co-associated with β catenin, top right; h lateral localisation of pneumococci (XY images, TIGR4 (green) and JAM-A (red)) with possible paracellular movement of 6B (green) co-associated with β catenin (red) (XZ images); i basal localisation of bacteria (23F, green) at the level of nuclei (blue) and insert pores N = 5 with replicates. Scale bar = 10 µm. j Scanning EM images of Detroit 562 cells infected with S. pneumoniae which appear as diplococci. Left—pneumococcal chain formation (strain 23F, arrow, scale bar = 4 µm); middle and right—micro-colony formation by strains 6B and TIGR4 on the epithelial surface (scale bar = 10 µm). k Micro-invasion of dPLY-TIGR4 shown by EM: left—scanning EM showing epithelial membrane folding (arrows) and pneumococci below the cell membrane surface (* scale bar = 2 µm); middle—internalisation of TIGR4 pneumococci encased within a vacuole (arrow, scale bar = 2 µm); and right—transmission EM showing transmigration of dPLY-TIGR4 pneumococci between epithelial cells (arrow, scale bar = 0.5 µm)