Figure 1. SarA functionally mimics part of the gp130 intracellular domain.

(A) The gp130 binding-of-STAT3 (GBS) sequence from S. Typhimurium SarA (amino acids 140-178) aligns with a conserved sequence in gp130 orthologues from human, cow, pig, mouse, chicken, and spotted gar fish. Amino acids are colored by type, and percent identity compared to SarA is listed for each ortholog. (B) SarA lacking the GBS is expressed but fails to drive STAT3-Y705 phosphorylation. HeLas were transfected with 5 μg pEGFP-Flag-sarA WT, Δ1-100 (deletion of SarA’s first 100 amino acids), or 139* (C-terminal truncation removing the GBS) 24 hrs before lysis and immunoblotting. (C) Domain diagrams of SarA-gp130 chimeras. (D) SarA-gp130 chimera drives STAT3 phosphorylation. STAT3 phosphorylation was measured 24 hrs after transfection in three experiments. (E, F) SarA-gp130 chimera under the endogenous promoter complements ΔsarA S Typhimurium induction of (E) STAT3 phosphorylation and (F) IL-10 in HeLa and LCL GM19154 by 24 hpi. No differences were noted in invasion or pyroptosis (Figure S1). IL-10 was measured by ELISA from three experiments. (G) SarA-gp130 chimera partially rescues ΔsarA virulence. Competitive index between Amp^R wild type (WT) and Kan^R strains was measured four days after an intra-peritoneal injection of 1,000 CFUs into C57BL/6J mice. From two experiments. One-sample t tests against zero (no change from 1:1 inoculum) on log₁₀-transformed data. (H) Gp130 ligands drive STAT3 phosphorylation in HeLa and LCL GM19154. Cells were treated with OSM (10 ng/mL) or IL-6 (50 ng/mL) for indicated time. (I) Dimeric gp130-SarA chimera induces more STAT3 phosphorylation than dimeric gp130. HeLa cells were transfected with the indicated constructs and STAT3 phosphorylation was assessed 24 hrs later. Unless noted, all p values are from Welch’s t test.