A single amino acid in the Salmonella effector SarA/SteE triggers supraphysiological activation of STAT3 for anti-inflammatory target gene expression

Abstract

Non-typhoidal Salmonella enterica cause an estimated 1 million cases of gastroenteritis annually in the United States. These serovars use secreted protein effectors to mimic and reprogram host cellular functions. We previously discovered that the secreted effector SarA (Salmonella anti-inflammatory response activator; also known as SteE) was required for increased intracellular replication of S. Typhimurium and production of the anti-inflammatory cytokine interleukin-10 (IL-10). SarA facilitates phosphorylation of STAT3 through a region of homology with the host cytokine receptor gp130. Here, we demonstrate that a single amino acid difference between SarA and gp130 is critical for the anti-inflammatory bias of SarA-STAT3 signaling. An isoleucine at the pY+1 position of the YxxQ motif in SarA (which binds the SH2 domain in STAT3) causes increased STAT3 phosphorylation and expression of anti-inflammatory target genes. This isoleucine, completely conserved in ~4000 Salmonella isolates, renders SarA a better substrate for tyrosine phosphorylation by GSK-3. GSK-3 is canonically a serine/threonine kinase that nonetheless undergoes tyrosine autophosphorylation at a motif that has an invariant isoleucine at the pY+1 position. Our results provide a molecular basis for how a Salmonella secreted effector achieves supraphysiological levels of STAT3 activation to control host genes during infection.

Keywords: IL-6; IL6ST; JAK-STAT; SOCS3; STM2585; Y705; gogC.

Figure 1:. SarA induces anti-inflammatory cell signaling and strong STAT3 phosphorylation.

(A) SarA upregulated genes significantly overlap with IL-10 target genes. Venn diagrams showing overlap of genes upregulated in a SarA-dependent manner (during S. Typhimurium infection in LCLs from ) and in an IL-10 or IL-6 dependent manner (after cytokine stimulation in dendritic cells from ). P-values obtained from a chi-squared test. (B) Wild-type S. Typhimurium infection leads to increased expression of anti-inflammatory genes. THP-1 cells were either stimulated with 10 ng/mL of IL-6 or infected with wild-type or ΔsarA S. Typhimurium. RNA was collected from cells 8 hrs post-infection and expression levels of SBNO2 and TNIP3 were measured via qPCR. Data are from three experiments. P-values obtained from a Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. (C) SarA GBS domain leads to greater STAT3 phosphorylation compared to gp130 GBS domain as quantified by western blot after overexpression in HeLa cells. Data are from four experiments. P-values obtained from unpaired t-tests with Welch’s correction comparing FLAG-SarA to FLAG-SarA:gp130 and FLAG-gp130 to FLAG-gp130:SarA. (D) SarA GBS domain leads to greater STAT3 phosphorylation compared to gp130 GBS domain as quantified by western blot after S. Typhimurium infection in THP-1 cells. Data are from four experiments. P-values obtained from a 2-way ANOVA with Tukey’s multiple comparisons test.

Figure 2:. Lack of SOCS3/SHP-2 binding does not explain supraphysiological activation of STAT3 by SarA.

(A) Diagrams of FLAG-SarA YxxV mutant constructs used in overexpression experiments. (B) Effects of mutations of SOCS3/SHP-2 binding motifs on phospho-STAT3. Constructs in (A) were overexpressed in HeLa cells. Mutating the YxxV SOCS3/SHP-2 binding motif had no significant impact on STAT3 phosphorylation as measured by western blot. Data are from three experiments. P-values obtained from a 2-way ANOVA with a Tukey’s multiple comparisons test. (C) Knockdown of SOCS3 and SHP-2 do not significantly increase SarA-induced STAT3 phosphorylation. HeLa cells were treated with either a non-targeting control (siGenome non-targeting #5), siSOCS3, or siSHP-2 48 hrs before infection with either a wild-type complemented or psarA:gp130 complemented strain of S. Typhimurium. STAT3 phosphorylation measured by western blot shows that knocking down negative regulators SOCS3 and SHP-2 were not able to restore psarA:gp130 induced activation to wild-type levels. Data are from four experiments. P-values obtained from a 2-way ANOVA with a Tukey’s multiple comparisons test.

Figure 3:. Isoleucine at pY+1 position leads to increased STAT3 binding and phosphorylation.

(A) Table of mutations made to YxxQ STAT3 binding motif in FLAG-SarA and FLAG-SarA:gp130 constructs. (B) Effects of mutants on STAT3 phosphorylation. Constructs in (A) were overexpressed in HeLa cells. STAT3 phosphorylation measured by western blot shows that mutating the pY+1 position in SarA from isoleucine to arginine leads to a significant decrease in STAT3 activation. Mutating the same position in SarA:gp130 from arginine to isoleucine restores STAT3 phosphorylation to wild type levels. Mutating the pY+2 position has no significant effect. Data are from three experiments. P-values are from a one-way ANOVA with Dunnett’s multiple comparisons tests comparing FLAG-SarA to its derived mutants and FLAG-SarA:gp130 to its derived mutants. (C) Isoleucine at pY+1 position leads to greater STAT3 phosphorylation during infection. THP-1 cells were infected with wild-type, psarA^I168R, psarA:gp130, or psarA:gp130^R168I complemented S. Typhimurium. STAT3 phosphorylation was measured by western blot. Data are from three experiments. P-values are from a 2-way ANOVA with Tukey’s multiple comparisons test. (D) FLAG-SarA constructs with arginine at pY+1 position bind less STAT3. FLAG-SarA, FLAG-SarA^I168R, FLAG-SarA:gp130, and FLAG-SarA:gp130^R168I were overexpressed in HeLa cells, followed by co-immunoprecipitation and probing for bound STAT3 via western blot. Data are representative of three experiments. (E) Mutating the pY+1 position has a minimal effect on STAT3 binding. Purified STAT3 was incubated with 25μM phosphopeptides and a thermal shift assay was used to calculate the melting temperature of the bound peptides as a proxy for binding affinity. Mutating the pY+1 position did not significantly affect melting temperature. Data are from two experiments. P-values are from a Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons test.

Figure 4:. Isoleucine at the pY+1 position makes SarA a better substrate for GSK-3 but has no effect on gp130.

(A) Isoleucine at the pY+1 position is required for SarA phosphorylation. Empty vector and panel of FLAG-SarA constructs were overexpressed in HeLa cells and co-immunoprecipitation confirms that while GSK-3 is bound to all constructs, tyrosine phosphorylation of SarA^I168R and SarA:gp130 was not detected. Data are representative of two experiments. (B) Mutation of isoleucine at the pY+1 position greatly reduces phosphorylation of SarA by GSK-3. GFP, GFP-SarAΔN44, and GFP-SarAΔN44^I168R were expressed in GSK-3α/β^−/−293ET cells, immunoprecipitated, and assessed for their ability to be tyrosine phosphorylated by GSK-3β in an in vitro kinase assay containing 1 mM ATP with or without recombinant Avi-GSK-3βS9A (0.2 μM) and His6-STAT3127-715 (0.2 μM). Data are representative of three experiments. (C) Isoleucine at the pY+1 position is not sufficient to induce greater levels of gp130-mediated STAT3 phosphorylation. Empty vector, FLAG-gp130, FLAG-gp130:sarA, and FLAG-gp130^R238I were overexpressed in HeLa cells for 24hrs, STAT3 phosphorylation assessed by western blot shows that the R238I single point mutation does not increase pSTAT3 levels compared to wild type gp130. Data are from four experiments. P-values obtained from a Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

Figure 5:. Isoleucine at pY+1 position promotes anti-inflammatory signaling bias.

(A) Arginine in the pY+1 position leads to decreased expression of anti-inflammatory genes during infection and isoleucine at this position rescues expression of these genes. THP-1 cells were infected with ΔsarA, wild-type complemented, psarA^I168R, psarA:gp130, or psarA:gp130^R1681 S. Typhimurium. RNA was collected from cells 8hrs post-infection and expression levels of SBNO2 and TNIP3 were measured via qPCR. Data are from three experiments. P-values obtained from a Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. (B) Arginine in the pY+1 position leads to decreased production of IL-10 during infection and is rescued with isoleucine at the position. THP-1 cells were infected with ΔsarA, wild-type complemented, psarA^I168R, psarA:gp130, or psarA:gp130^R168I S. Typhimurium. Cell supernatant was collected at 8 hrs and 24 hrs post-infection and IL-10 was measured via ELISA. Data are from three experiments. P-values obtained from a two-way ANOVA with a Sidak’s multiple comparison test.

Figure 6:. SarA is variably present in Salmonella serovars but isoleucine at pY+1 position is always conserved.

(A) SarA is present in diverse S. enterica subspecies and serovars. Mean E-values (indicated by color) from BLASTp of SarA in 21,223 Salmonella isolates were merged with an existing whole genome maximum-likelihood phylogeny (Worley et al., 2018). Serovars without corresponding BLASTp data are in gray. (B) Isoleucine at pY+1 is invariable across Salmonella isolates that contain SarA and homologs of the human protein GSK-3β. Sequence logos were derived from multiple sequence alignment and reflect the relative frequency of each residue across all aligned sequences. The YxxQ motif that is homologous between SarA and IL6ST is highlighted, as well as the known autophosphorylation site in the kinase GSK-3β. Known chemical classification of each residue is indicated by color.