Fig. 2.

Effect of β-hexosaminidase on intracellular growth of Mm. (A) Flow cytometry histogram of S2 cells infected by MmGFP 48 h after initiation of infection. The fluorescence of Mm was greater in HEXO2 RNAi-treated S2 cells, despite equivalent initial uptake. Histogram is representative of three independent experiments. (B) Growth curve of Mm in WT or HexB^−/− BMDM. Mm shows increased intracellular growth in HexB^−/− BMDM. Activated BMDM (by treatment with IFN-γ and LPS) did not support growth of the bacilli. The graph shown is normalized for bacterial uptake (after washing of monolayer) and representative of three independent experiments. (C) MmGFP-infected HexB^−/− BMDM were stained with Lysotracker red 24 h after initial bacterial uptake. (Upper) Uptake of live bacilli. (Lower) Uptake of heat-killed bacilli. Fusion of phagosomes containing heat-killed Mm with lysosomes is indicated by overlap of the entire bacterium with red fluorescence, demonstrating the low pH (5.5) of the phagosome. Examples of bacteria are indicated by arrows.