Fig. 3 . M. smegmatis overexpressing Mtb LpdC retains coronin-1 on its phagosome and survives better within the macrophage. (A) BCG, M. smegmatis (Msmeg) and recombinant M. smegmatis expressing Mtb LpdC (rMsmeg) grown to lag, log and stationary phase in 7H9-OADC were pelleted and cultured (16-18 hours for BCG and 3-4 hours for M. smegmatis) in modified Dubos medium. CFPs corresponding to 108 CFU were 10% TCA precipitated and the culture pellets were lysed by sonication in 75 mM Tris pH 8.8 plus 4 mM EDTA and 100 mM NaCl. CFPs and soluble fractions of bacterial lysates were resolved by SDS-PAGE followed by immunoblotting with anti-LpdC antibodies. (B) J774 cells adhering to glass coverslips were infected with GFP-BCG, GFP-smegmatis (Msmeg) or recombinant M. smegmatis expressing Mtb LpdC and GFP (rMsmeg). At 4 hours post-phagocytosis, cells were fixed, permeabilized and stained with rabbit anti-coronin-1 as described previously (Sendide et al., 2005a). Samples were analyzed by digital confocal microscopy using an epifluorescence microscope. The yellow signal indicates colocalization of green (bacteria) and red (coronin) fluorescence. (C) Quantification of bacterial phagosomes surrounded with coronin-1 observed in 15-20 cells from three individual experiments. (D) Macrophages were infected with the indicated bacteria and at 1 and 4 hours post-phagocytosis, bacteria were released by treatment with 0.1% Triton X-100 in PBS. Thereafter serial dilutions were prepared in PBS and plated on 7H11 plates in triplicate as described previously (Sendide et al., 2004). The data are presented as mean ± s.d. of CFU counts obtained from two independent experiments.