Figure 7. M.tb localizes to LC3-positive compartments. (A) GFPLC3expressing macrophages were infected with mCherry-M.tb (MOI 50
or 10, 6 h) and then fixed and observed by confocal microscopy. At high MOI there are GFP-LC3 rings surrounding (arrowhead) around mCherry-M.tb and co-localization indicated by yellow pixels (arrow) that are not seen in macrophages challenged at low MOI. (B) LAMP1 immunostaining of GFP-LC3 cell line macrophages infected with M.tb Edrman (MOI 50 or 10, 5 h) demonstrates co-localization of LC3 and lysosomal markers (arrowheads).
Figure 2. Differences in expression of rv2390c9::GFP in Mtb present in vaccinated versus mock-treated mice. Erdman(rv2390c9::GFP, smyc9::mCherry) was inoculated into vaccinated or mock-treated C57BL/6J WT mice for up to 56 days. (A) shows 3D confocal images from a 14, 28, or 42 day infection. All bacteria are marked in red (smyc9::mCherry), reporter signal is shown in green (rv2390c9::GFP), nuclei are marked in grayscale (DAPI), and phalloidin staining of f-actin is shown in blue. Scale bar 10 mm. (B) shows quantification of the GFP/mm3 signal for each bacterium measured from multiple 3D confocal images, at the indicated time points. Each point on the graph represents a bacterium or a tightly clustered group of bacteria (mock-treated – filled symbols, vaccinated – open symbols). Horizontal lines mark the median value for each group. p-values were obtained with a Mann-Whitney statistical test.