Fig. 3.

Cholesterol utilization is required for mycobacterial persistence in chronically infected mice and replication of IFN-γ-activated macrophages. (A) The abilities of wild-type and Δmce4 mutant bacteria to persist in mouse lungs were compared. C57BL/6 mice were infected i.v. with a 1:1 mixture of wild-type (H37Rv) and Δmce4 mutant bacteria, and the abundance of each strain in lung homogenates was quantified at the indicated times by plating. (B) The abilities of wild-type and mce mutants to replicate in bone marrow-derived macrophages (BMM) were compared. The number of cfu recovered from cell lysates at 0 and 5 days after infection are presented. The indicated macrophages were stimulated with IFN-γ 24 before infection. The data in A and B are representative of three or four independent experiments, respectively. Error bars indicate standard deviation. (C–F) Colocalization of cholesterol and M. tuberculosis in IFN-γ-activated macrophages. Filipin-stained cholesterol (C) and RFP-expressing bacteria (D) were simultaneously visualized in infected BMMs by using confocal microscopy. These images were merged to demonstrate colocalization (E, orange) and overlaid on a DIC image (F). In representative optical sections, 53–85% of RFP-positive pixels colocalized with filipin. The three fluorescent images shown represent a single optical section.