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. Author manuscript; available in PMC: 2019 Jan 15.
Published in final edited form as: J Immunol. 2017 Dec 4;200(2):607–622. doi: 10.4049/jimmunol.1700725

Figure 4. Deletion of the Brucella guanylate cyclase reduces cellular cyclic di-GMP levels and IFN-β expression.

Figure 4

(A) MEFs from wild-type (WT) mice were transfected with dsDNA90 (3μg/ml) or cGAMP (1μg/well) or infected with Brucella abortus S2308 or Brucella abortus Δ1520 (MOI 1000:1) for 4 hrs, fixed and subjected to immunofluorescence microscopy analysis of STING. Pronounced translocation of STING was observed as aggregated speck formation in the perinuclear region 4 hrs after cells were transfected with dsDNA90, cGAMP or infected with Brucella abortus S2308, but not after infection with Brucella abortus Δ1520. Antibody staining is shown in middle panels for STING and nuclei staining (DAPI) is shown in blue on the right panels. Left panels are merged images from those shown on middle and right panels including staining with anti-Brucella LPS in green. Data are representative of three independent experiments. Size bar shown corresponds to 25 μm in all panels. (B) Wild-type Brucella or Δ1520 mutant strains were transfected with a c-di-GMP regulated lux reporter system. Bacteria were grown to stationary phase and relative luminescence (RLU) units measured (n=4 replicates). Significant differences between wild-type virulent Brucella compared to Δ1520 mutant is denoted by an asterisk (p<0.05). Macrophages derived from C57BL/6 and STING−/− mice were infected with B. abortus wild-type or Δ1520 mutant strain (MOI 100:1). After 17 hrs, total RNA was purified and the gene expression of IFN-β (C) was analyzed by qPCR and IL-1β (D) was measured in the supernatant by ELISA. (E) BMDMs from wild-type mice were treated or not-treated with Ebselen. Cells were either not-infected (medium) or infected (MOI 100:1) with B. abortus for 24 hrs with or without Ebselen (50μM). Supernatants were then harvested and IFN-β production measured by ELISA. (F) BMDMs from C57BL/6 and STING−/− mice were infected with B. abortus wild-type or Δ1520 mutant strain (MOI 10:1) for 48 hrs and bacterial CFU counts were analyzed. Data are representative of three independent experiments and three replicates in each experimental group. Significant differences comparing C57BL/6 mice versus STING KO is denoted by an asterisk (*) and between wild-type Brucella strain 2308 versus Δ1520 mutant is denoted by an $ (two-way ANOVA, p< 0.05).