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. 2023 Mar 9;120(11):e2217602120. doi: 10.1073/pnas.2217602120

Fig. 1.

Fig. 1.

C. burnetii inhibits RIG-I signaling by a Dot/Icm-dependent mechanism. (A) qPCR analysis of C. burnetii genome equivalents (GE) during infection of PMA-differentiated THP-1 pretreated with IFN-β or untreated infected at an MOI of 20. Data are mean ± SD of three independent experiments. P < 0.05 untreated vs. IFN-β at D3 to D7 by unpaired t test. (B) ISRE reporter activation by C. burnetii infection. Human Embryonic Kidney 293 (HEK293) ISRE-luciferase cells were infected for 48 h with C. burnetii at an MOI of 200 or transfected with poly(I:C). Data are mean ± SD of three technical replicates representative of two independent experiments. P < 0.01 uninfected vs. poly(I:C) by one-way ANOVA with Dunnett’s post hoc test. (C) ISRE reporter induction by poly(I:C). Cells were infected for 24 h with indicated strain at an MOI of 200, transfected with poly(I:C), and incubated an additional 24 h before luminescence was measured. Data are mean ± SD of five technical replicates representative of three independent experiments. P < 0.01 wild type (WT) Cb vs. uninfected and icmL::Tn, uninfected vs. icmL::Tn n.s., by one-way ANOVA with Tukey’s post hoc test. (D) ISRE reporter induction by cGAS-STING pathway activation. Cells were infected for 24 h with indicated strain at an MOI of 200, transfected with plasmids encoding cGAS and stimulator of Interferon genes (STING), and incubated an additional 24 h before luminescence was measured. Data are mean ± SD of two independent experiments. n.s. for uninfected vs. WT and dotA::Tn by one-way ANOVA with Tukey’s post hoc test. (E) qPCR analysis of interferon Beta gene (IFNB) messenger RNA (mRNA) in HEK293T RIG-I (DDX58) WT and KO cells. Cells were infected for 24 h with wild-type Cb at an MOI of 1,000, transfected with poly(I:C) and incubated an additional 24 h. Data are relative to uninfected DDX58+/+ without poly(I:C) and normalized to 18S. Data are mean ± SD of two independent experiments. P < 0.01 poly(I:C) + no Cb vs. all other conditions; all other comparisons, n.s. by two-way ANOVA with Tukey’s post hoc test. (F) Screen for C. burnetii transposon insertion mutants unable to block ISRE-luciferase production by poly(I:C). HEK293-ISRE cells were infected at an multiplicity of infection (MOI) of 200 for 24 h before transfection with poly(I:C) for 24 h. Data are mean ± SD of three independent experiments. P < 0.0001 for WT vs. icmL::Tn, cbu1387::Tn and cbu2013::Tn; WT vs. all others n.s. by one-way ANOVA with Dunnett post hoc test. (G) ISRE reporter induction by poly(I:C) in cells infected with indicated C. burnetii mutant. Data are mean ± SD of five replicates representative of two independent experiments. P < 0.01 for WT vs. cbu1387::Tn, cbu2013::Tn, icmL::Tn and uninfected; WT vs. cbu1752::Tn n.s. by one-way ANOVA with Tukey’s post hoc test. (H) ISRE reporter induction by poly(I:C) in cells infected with indicated C. burnetii mutant or complemented strain. Data are mean ± SD of 10 replicates representative of two independent experiments. P < 0.01 for cbu1387::Tn vs. cbu1387::Tn complement and cbu2013::Tn mutant vs. cbu2013::Tn complement by one-way ANOVA with Šídák’s post hoc test. (I) qPCR analysis of cytokine mRNA in J774A.1 cells infected with indicated C. burnetii strain for 4 d at an MOI of 500. Results are relative to those of uninfected J774A.1. P < 0.05 for WT vs. emcB::Tn and emcA::Tn Ifnb1; WT vs. emcB::Tn Il6 n.s., WT vs. emcA::Tn Il6 <0.05 by one-way ANOVA with Dunnett’s post hoc test.