Figure 2. The type IV secreted effector SdhA suppresses induction of interferon by L. pneumophila.

(A) The 11C11 mutant harbors a transposon insertion in sdhA. The transposon insertion site is in the 3′ end of the open reading frame of the sdhA locus at nucleotide position 3421. (B) A clean deletion mutant of sdhA recapitulates the 11C11 transposon mutant and hyperinduces type I interferon. Bone marrow derived Myd88 ^−/− Trif ^−/− macrophages were infected with stationary phase L. pneumophila strains at a MOI of 1. Cell supernatants were harvested 8 hours post infection and assayed for type I interferon induction by an L929-ISRE luciferase bioassay. Type I interferon levels were determined by generating a standard curve with recombinant IFNβ An unmarked clean deletion of sdhA was compared to wild type, Δdot, the transposon mutant 11C11, and a triple deletion of sdhA and the two L. pneumophila paralogs, sidH and sdhB. Differences in IFNβ induction were statistically significant between WT L. pneumophila and the transposon mutant 11C11 (***, p<0.0005, Student's t-test). Differences between 11C11, ΔsdhA and ΔsdhAΔsdhBΔsidH were not statistically significant (ns, p>0.05, Student's t-test). (C) A clean deletion mutant of sdhA recapitulates the 11C11 transposon mutant and hyperinduces transcriptional activation of Ifnb. Bone marrow derived Myd88 ^−/− Trif ^−/− macrophages were infected with wild type, Δdot, ΔsdhA, 11C11, ΔsdhAΔsdhBΔsidH stationary phase L. pneumophila and transcriptional induction of Ifnb was analyzed by quantitative RT-PCR. (D) Complementation of the sdhA mutant results in loss of the Ifnb hyperinduction phenotype. MyD88 ^−/− Trif ^−/− BMDM were infected at an MOI of 1 with ΔflaA, Δdot, ΔflaAΔsdhA and ΔflaAΔsdhA L. pneumophila carrying vector or a plasmid expressing full length SdhA. Expression of Ifnb message was assessed by quantitative RT-PCR 4 hours post infection.