Fig. 3.

Phagosomal escape, but not bacterial protein synthesis or viability, is required for SchuS4-mediated reduction of IL-1β, IL-6 and CXCL1 mRNA. a-c BMM were primed with P3C (500 ng/ml) and treated with cytochalasin D (CytoD) to inhibit bacterial phagocytosis or bafilomycin (Bafilo) to inhibit bacterial escape from endosomes prior to infection, where indicated. BMM were infected with SchuS4 MOI 50 or SchuS4 ΔfevR MOI 50. Mock-infected cells served as negative controls. a RNA was harvested 5 h after infection. qRT-PCR was performed to examine the quantity of the indicated transcripts, normalized to GAPDH. * p < 0.05 compared to the indicated samples; n.s. = not significant compared to the indicated samples. b WT or TLR2^−/− BMM were primed with monophosphoryl lipid A (25 ng/ml) and infected with SchuS4 MOI 50, F. novicida MOI 10 or coinfected with both SchuS4 MOI 50 and F. novicida MOI 10. Secretion of IL-1β, IL-6 and CXCL1 was assessed by ELISA. * p < 0.05: F. novicida-infected compared to F. novicida/SchuS4-coinfected cells. c Cytoplasmic bacteria were quantified 5 h after infection using a phagosomal-integrity assay. * p < 0.05 compared to WT SchuS4 infection of untreated cells. d, e BMM were primed with P3C (500 ng/ml) and infected with MOI 50 of SchuS4, SchuS4 pretreated with chloramphenicol (Chloramp) to inhibit bacterial protein synthesis, or inoculated with SchuS4 killed by treatment with PFA or irradiation (Irradiate). d Cytoplasmic bacteria were quantified 5 h after infection using a phagosomal-integrity assay. * p < 0.05 compared to untreated SchuS4. e RNA was harvested 5 h after infection. qRT-PCR was performed to examine the quantity of the indicated transcripts, normalized to GAPDH. * p < 0.05 compared to uninfected cells. Each data point depicts the mean of triplicate samples. Error bars represent SEM. Data are representative of 3 independent experiments.