FIG. 5.

Analysis of phagosome trafficking in macrophages infected with Y. pseudotuberculosis or Y. pestis by anti-cathepsin D staining and confocal microscopy. IP2666c/GFP, IP2666cphoPΔ/GFP, KIM10⁺/GFP, and KIM10⁺phoPΔ/GFP were grown in the presence of IPTG and used to infect J774A.1 macrophages at an MOI of 10 for Y. pseudotuberculosis or 2 for Y. pestis. At different times postinfection the cells were fixed and stained with anti-cathepsin D antibody and a secondary antibody conjugated to Alexa 594. The samples were analyzed by confocal microscopy. (A and D) Representative images of the merged anti-cathepsin D and GFP signals obtained with cells infected for 8 h with IP2666cphoPΔ/GFP (A) or for 1.5 h with KIM10⁺phoPΔ/GFP (D). The arrows indicate cathepsin D-positive phagosomes. (Insets) Enlargement of the region indicated by the white rectangle. The left inset shows merged GFP and cathepsin D signals, and the right inset shows cathepsin D signals alone. (B) Percentages of cathepsin D-positive phagosomes in macrophages after 8 h of infection with IP2666c/GFP or IP2666cphoPΔ/GFP calculated by using merged images captured at a magnification of ×40. (E) Percentages of cathepsin D-positive phagosomes in macrophages containing KIM10⁺/GFP or KIM10⁺phoPΔ/GFP at 1.5 h postinfection determined in the same manner. The data in panels B and E are the averages of three independent experiments. The error bars indicate the standard deviations. (C and F) Numbers of CFU recovered from J774A.1 macrophages infected with IP2666c/GFP or KIM10⁺/GFP or the corresponding phoP mutants at the times indicated. Each data point represents the average ± standard deviation of the values recovered from triplicate wells. The results are representative of two independent experiments.