FIG 3.

Confocal microscopy of internalized M. tuberculosis bacilli and bacterial replication in neurons. HT22 cells (A to C), Neuro-2a cells (D to F), and primary neurons (G to I), established from the hippocampi of 17-day-old C57BL/6 mouse embryos, and BV2 cells (J to L) were infected with M. tuberculosis for 24 h and then subjected to immunohistochemistry for 6 h, 24 h, and 48 h, and the numbers of CFU were determined from lysed cultures (M). The fluorescence images of phalloidin-labeled cell lines, including HT22, Neuro-2a, and BV2, and MAP2-labeled primary neurons highlight the cytoskeletal proteins (red) to demonstrate the cytosolic location of GFP-expressing bacilli (green). The internalized bacilli can be seen in the top and side images in the orthogonal views, which show the colocalization of cytoskeleton and bacilli in the x-y plane of an optical section of the z-stack. Phase-contrast images of cultured cells are presented in panels A, D, G, and J. (M) Bacterial replication in HT22 and Neuro-2a cells and primary neurons assessed at 6 h, 24 h, and 48 h. The results are the means and SD of quadruple experimental data sets and are representative of one of three similar experiments.