The Salmonella Effector Protein SopA Modulates Innate Immune Responses by Targeting TRIM E3 Ligase Family Members (original) (raw)
Fig 5
SopA enhances the ability of TRIM56 and TRIM65 to stimulate interferon-β expression.
(A-B) HEK293T cells were co-transfected with plasmids expressing either wild type SopA or its catalytic mutant SopAC753S along with plasmids expressing TRIM65 (25 ng), TRIM56 (10 ng), MDA5 (50ng) or RIG-I(2CARD) (4ng) as indicated, and reporter plasmids expressing firefly luciferase under the control of interferon-β promoter and renilla luciferase (to standardize the transfection efficiency). The activity of the interferon-β promoter was analyzed 18 hs after transfection with the Dual Luciferase Reporter Assay System (Promega). Values represent the mean +/- standard deviation of the relative levels of normalized firefly luciferase from 3 independent experiments. The normalized firefly luciferase activity relative to mock-transfected cells was 7.3 ± 3.9 for MDA5 only transfected cell (B) and 17.8 ± 9.7 for RIG-I(2CARD) only transfected cells. *: indicates statistically significant difference (p < 0.05) (C) Primary MEFs were infected with wild type S. Typhimurium, the ∆sopA or the effectorless (∆sipA ∆sptP ∆avrA ∆sopE ∆sopE2 ∆sopA ∆sopB sopD ∆sopD2 ∆slrP) isogenic mutant strain and the levels of interferon-β gene expression were measured by qRT-PCR 4 and 8 hours after infection. Data represent the mean ± standard deviation of the _n_-fold expression of interferon-β mRNA over GAPDH relative to non-infected cells. This experiment was repeated 3 times with equivalent results. (D) Primary MEFs were infected with the effectorless S. Typhimurium strain (∆sipA ∆sptP ∆avrA ∆sopE ∆sopE2 ∆sopA ∆sopB sopD ∆sopD2 ∆slrP) or the effectorless strain expressing plasmid-born wild type SopA, its catalytic mutant SopAC753S, or an irrelevant effector. The levels of interferon-β gene expression were measured by qRT-PCR 8 hs after infection. Data represent the mean ± standard deviation of the _n_-fold expression of interferon-β mRNA over GAPDH relative to non-infected cells. The results of a representative experiment (out of 5 independent experiments) is shown. The p value for the difference in interferon-β mRNA levels between MEFs infected with the Δeffectors (pSopAwt) strain or Δeffectors (pSopAC753S) was p = 0.002 (n = 5). (E) Immortalized (MEF wt) and TRIM56-deficient (MEF TRIM56 KO) murine embryonic fibroblasts were infected with wild type S. Typhimurium or the effectorless (∆sipA ∆sptP ∆avrA ∆sopE ∆sopE2 ∆sopA ∆sopB sopD ∆sopD2 ∆slrP) isogenic mutant strain expressing plasmid-born wild type SopA or its catalytic mutant SopAC753S. The levels of interferon-β gene expression were measured by qRT-PCR 8 hours after infection. Data represent the mean ± standard deviation of the _n_-fold expression of interferon-β mRNA over GAPDH relative to non-infected cells. A representative experiment out of 7 independent experiments is shown. The p values for difference between the interferon-β mRNA levels in wild type MEFs infected with the Δeffectors (pSopAwt) or Δeffectors (pSopAC753S) S Typhimurium strains was p = 0.035 while the difference in TRIM56 KO cells was p = 0.639 (n = 7 in each category). (F) Effect of SopA in S. Typhimurium stimulation of pro-inflammatory cytokine expression in the mouse intestine. C57/BL6 nramp+/+ mice were orally infected with the effectorless S. Typhimurium strain (ΔsipA ΔsptP ΔavrA ΔsopE ΔsopE2 ΔsopA ΔsopB sopD ΔsopD2 ΔslrP) (n = 6) or the effectorless strain expressing plasmid-born wild type SopA (n = 6). Seventy-two hours after infection the relative levels of the indicated cytokines in the intestine were measured by qRT-PCR. Data were normalized to the levels of GAPDH and represent the mean ± standard error relative to uninfected control animals (n = 3). *: indicates statistically significant difference (p < 0.05).