Figure 8.

TetR-controlled GFP expression in intraphagosomal M.tuberculosis H37Rv. Murine bone marrow-derived macrophages grown on coverslips in 24 well plates were infected with live bacteria at an MOI of 5–10 bacteria: 1 macrophage. Four hours post infection, the macrophage monolayers were washed three times with warm PBS, followed by the addition of complete tissue culture medium containing 100 μg/ml amikacin to kill extracellular bacteria. The monolayers were washed again 8 h post infection. Twenty-four hours post infection, 100 ng/ml ATc were added to wells shown in (C) and 72 h later the coverslips were analyzed by microscopy as described in Materials and Methods. The left panels depict the cell monolayers in phase contrast and the right panels show the corresponding fluorescence image. Macrophages were infected with M.tuberculosis transformed with P_myc1tetO-GFP (A); P_myc1tetO-GFP + P_imyc-tetR (B and C). (D) Cells that are infected with Mtb lacking GFP and treated with ATc. Samples were prepared on a different day than those shown in (A–C), but the images were acquired under the exact same conditions. The relative brightness of control samples was similar on the two different days (insert: macrophages infected with Mtb constitutively expressing GFP).