FIG. 7.

M. tuberculosis phagosomes in HeLa-Rab7 cells stain positively for Rab7 but exclude LAMP-1. HeLa-Rab7 cells were maintained and expanded in the presence of tetracycline (5 μg/ml). One day prior to infection with M. tuberculosis, tetracycline was removed from the culture medium to induce Rab7 expression. The HeLa cells were incubated for 2 h with M. tuberculosis using a multiplicity of infection of 50:1. Nonadherent bacteria were washed away, and the monolayers were incubated for 2 additional days. Monolayers were fixed and processed for cryoimmunoelectron microscopy. Rab7 was stained with 15-nm immunogold particles (large arrowheads) and is abundant on the M. tuberculosis phagosomal membranes. Mycobacterial lipoarabinomannan was stained with 5-nm gold particles and is present on the mycobacterial cell wall (small arrows). LAMP-1 immunogold staining (10-nm gold particles) is scarce on the M. tuberculosis phagosomal membrane but is present on adjacent cytoplasmic vesicles (small arrowheads). Magnification, ×33,930.