Figure 6

The germination state of spores influences the viability of intracellular B. anthracis. RAW264.7 cells were incubated for 30 min with dormant B. anthracis spores (MOI 10) in DMEM in the presence (+, black bars) or absence (-, white bars) of FBS (10%), or, with pre-germinated spores (MOI 10) in DMEM in the absence of FBS (grey bars). B. anthracis spores were pre-germinated by incubation for 30 min in PBS pH 7.2 supplemented with L-alanine and L-inosine (both at 10 mM), and then washed twice with PBS pH 7.2 to remove germinants. After 30 min, the cells were washed to remove extracellular B. anthracis, and then further incubated with FBS (10%) and, as described under "Methods," with gentamicin to germinate and kill any remaining spores that had not been germinated. After 15 min, the cells were washed and then further incubated in the absence of gentamicin. At 5, 60, or 240 min after removal of gentamicin, as indicated, the RAW264.7 cells were lysed, and the lysates were evaluated for viable B. anthracis, as described under Materials and Methods. The data were rendered as the fold-change in recoverable CFU in the absence or presence of FBS, relative to cells at 5 min post infection in the absence of FBS. The rendered data were combined from three independent experiments, each conducted in triplicate. Error bars indicate standard deviations. The P values were calculated to evaluate the statistical significance of the differences in recoverable CFU between cells infected in the absence or presence of FBS.