FIG 4.

Replication-deficient mutant SCHU S4 ΔpurMCD induces wild-type levels of C3-dependent macrophage death. MDM were infected with WT or ΔpurMCD SCHU S4 at a low MOI in the presence of HS or at a high MOI in the presence of C3-depleted serum. The results shown are averages from 3 independent experiments. The average MOI for WT was 22 in HS and 890 in C3-depleted HS. The average MOI for SCHU S4 ΔpurMCD was 17 in HS and 672 in C3-depleted HS. (A) *, significant differences in bacterial burden compared to the WT in HS or significant differences in LDH release compared to uninfected MDM at each time point. ns, not significant. (B to F) MDM infected in HS were stained with Zombie Red (red) and rabbit antiserum to Francisella (green) and then mounted in Prolong Gold with DAPI (blue). Individual MDM infected with either WT or ΔpurMCD SCHU S4 were enumerated based on their bacterial load and incidence of Zombie Red-positive staining. (B and C) Relative frequency distributions for bacterial load for 3 h p.i. (B) and 24 h p.i. (C). *, significant differences between the percentage of WT SCHU S4- and SCHU S4 ΔpurMCD-infected macrophages containing the indicated range of bacterial counts. (D) Representative images of Zombie Red-stained MDM infected with either WT SCHU S4 or SCHU S4 ΔpurMCD in the presence of HS. Scale bars, 20 μm. (E) Percentage of Zombie Red-positive cells found at 24 h p.i. under each infection condition. *, significant differences compared to uninfected MDM. ns, not significant. (F) There were no significant differences between the percentages of Zombie Red-positive cells in WT SCHU S4- and SCHU S4 ΔpurMCD-infected MDM found in each bacterial load category at 24 h p.i.