The Salmonella Effector SarA Activates STAT3 to Promote an Anti-Inflammatory Response and Increase Virulence

Abstract

Salmonella enterica is an important foodborne pathogen that utilizes secreted effector proteins to manipulate host pathways to facilitate survival and dissemination. Different S. enterica serovars cause disease syndromes ranging from gastroenteritis to typhoid fever and vary in their effector repertoire. Knowledge of this effector repertoire is still incomplete. The effector protein encoded by stm2585 had previously been identified as relevant to in vivo infection, but the mechanism of action was unknown. We leveraged S. enterica’s natural genetic diversity to identify stm2585, here designated sarA (Salmonella anti-inflammatory response activator), as a Salmonella effector that induces production of the anti-inflammatory cytokine IL-10 in lymphoblastoid cell lines (LCLs) through activating STAT3 to promote virulence. RNA-seq of cells infected with either ∆sarA or wild-type S. Typhimurium revealed that SarA activated STAT3 transcriptional targets. Consistent with this, SarA was necessary and sufficient for STAT3 phosphorylation, STAT3 inhibition blocked IL-10 production, and physical interaction of SarA and STAT3 was identified by co-immunoprecipitation. These effects of SarA contributed to intracellular replication in vitro and bacterial load at systemic sites in mice.In order to further characterize SarA’s mechanism, we searched for interactions with host proteins. A yeast-two-hybrid screen revealed that SarA bound the cytoplasmic region of IL-7Rα. Co-expression of FLAG-SarA and IL-7Rα in HeLas resulted in phosphorylation of IL-7Rα and interaction of IL-7Rα with FLAG-SarA, as confirmed by co-immunoprecipitation of FLAG-SarA. In LCLs, siRNA against IL-7Rα moderately inhibited SarA’s ability to induce IL-10. However, IL-7Rα’s endogenous ligands IL-7 and TSLP did not mimic SarA’s effect, and IL-7Rα knockout mice did not provide ΔsarA with any benefit versus wild-type in competitive index, so IL-7Rα’s contribution to SarA’s activation of STAT3 is still unclear. Mass spectrometry of proteins which co-immunoprecipitated with SarA confirmed interaction with STAT3 and revealed several other interacting proteins. Mass spectrometry also indicated that SarA contained phosphorylated residues, including a phosphorylated tyrosine in a previously reported STAT3 binding motif. Mutation of this phosphorylated tyrosine to phenylalanine on a bacterial expression plasmid greatly reduced SarA’s ability to induce IL-10 in LCLs. The same mutant on a mammalian expression plasmid showed less activation and no co-immunoprecipitation of STAT3. This indicated that SarA’s interaction with STAT3 was at least partially dependent on that tyrosine, and suggested that SarA was directly binding STAT3 at this site.Our results demonstrate the power of using comparative genomics for identifying effectors and that S. enterica has evolved mechanisms for activating an important anti-inflammatory pathway. We also characterize SarA as a potential basis for immunomodulatory therapy.