Antigen presence in the cytosol. (A–D) Bone marrow macrophages infected for 24 hours with ΔureC hly⁺ rBCG (A and B) and parental BCG (C and D). The images are projections of 2 confocal planes from specimens stained for BCG (green), F-actin (red), and DNA (blue) (overlay in A and C). Material stained with an antibody raised against BCG is dispersed throughout the cytoplasm in cells infected with ΔureC hly⁺ rBCG while BCG material is exclusively located in bacteria within cells infected with parental BCG. Scale bar in A–D: 10 μm. (E and F) Fine structural analysis of mycobacterial localization in bone marrow macrophages 24 hours after infection with ΔureC hly⁺ rBCG (E) and parental BCG (F). Panel E shows strong staining of a bacterium in a phagosome; additional staining for mycobacterial material was found in the cytoplasm (indicated by an arrow). However, cytoplasmic staining was greatly reduced in cells infected with parental BCG (F). Quantification of the density of immunogold signal in the cytoplasm of infected cells: parental BCG, 3.5 ± 2.3 gold particles/μm²; ΔureC hly⁺ rBCG, 7.0 ± 3.7 gold particles/μm²; P = 0.022 (Mann-Whitney U test). Scale bar in E and F: 1 μm.