Figure 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. ns = not significant compared to the indicated samples. (B) Wild type (WT) or TLR2^−/− BMM were primed with MPLA (25 ng/mL) and infected with SchuS4 MOI 50, F. novicida MOI 10, or co-infected 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 only infected compared to F. novicida/SchuS4 co-infected 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 three independent experiments.