FIG 7.

Transit through autophagosome-related compartments increases the interaction of B. abortus with new host cells. HeLa epithelial cells were infected with B. abortus 2308 at a MOI of 500 for 34 h and were then either treated with 10 mM 3-methyladenine (3-MA), an autophagosome inhibitor, or left untreated (–) for 14 h. Intracellular bacteria were then extracted, purified (Int), and used in a new round of infection on HeLa epithelial cells at a MOI of 10. After 2 h of incubation, cells were extensively washed and processed by differential intracellular/extracellular immunofluorescence as described for Fig. 2. (A) Percentage of cells with associated bacteria; (B) mean number of bacteria per infected cell; (C) percentage of internalized Brucella bacteria; (D) number of intracellular bacteria in 100 cells. (E) HeLa epithelial cells were infected and treated as described above. At 2 h postinfection, cells were lysed, and intracellular bacteria were quantitated by plate counting. Each value is the average of at least three independent determinations. Statistical significance was calculated by Student’s t test. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.005) from intracellular bacteria that were left untreated.