FIG 1 .

SpyA-deficient GAS bacteria evade macrophage killing. To measure total and intracellular killing, BMDMs isolated from C57BL/6 mice were activated through overnight incubation in DMEM supplemented with 2% FBS. (A) Total killing was measured by recovering total CFU of cells infected with GAS (AP) at an MOI of ~10 after 2 and 4 h. (B) Intracellular killing was assessed by recovering CFU from cells infected with GAS for 30 min followed by 100 µg/ml of gentamicin treatment (1 h); levels of internalized bacteria were monitored over 2 and 4 h in serum-free media. (C) BMDMs were infected with S. aureus RN4220 at an MOI of ~5, and intracellular killing was monitored at 4 and 6 h. (D) Transfected J774 cells were infected with ΔspyA GAS for 2 h in 2% FBS media for a total killing assay. Data shown are representative of the results of multiple repeats. Error bar; SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. = not significant; n = 3 (Student’s two-tailed unpaired t test or one-way ANOVA and Tukey’s multiple-comparison test).