FIGURE 4.

Intracellular growth is restored to the Δicl1 Mtb through the induction of oleate-containing lipid droplets in the infected cell. The survival of the Δicl1 mutant is impaired in macrophages; however, growth could be restored by preloading the host cells with exogenous oleate or by adding VitB12 to the cell culture medium to facilitate operation of the MMP. A, induction of macrophage lipids droplets, detected with BODIPY 493/503 (green), Δicl1 strain expressing pVV16-mCherry (smyc′::mCherry) (red), and nuclei (blue) in oleate-loaded macrophages versus untreated macrophages over the duration of the experimental period. B, bacterial cfu counts were determined at 2-day intervals across an 8-day period for the Δicl1 Mtb in untreated, control macrophages (triangle), in lipid droplet-containing, oleate-loaded macrophages (circle), and in macrophage supplemented by the addition of VitB12 to the medium (square). C, the experiment was repeated with a Δicl1 strain expressing pVV16-mCherry (smyc′::mCherry), and bacterial proliferation was quantified by measuring bacterial mCherry fluorescence across an 8-day infection period. Both methods demonstrated the enhanced growth and survival of the Δicl1 mutant in the presence of either oleate-induced droplets or exogenous VitB12. Error bars, Standard Deviation values from three representative replicates.