Tumor Necrosis Factor Receptor-Associated Factor 6(TRAF6)Mediates STAT3 Activation Upon Salmonella Typhimurium Infection

Salmonella enterica serovar Typhimurium(S.Typhimurium)can inject effector proteins into host cells via Type III secretion systems(T3SSs).These effector proteins modulate a variety of host transcriptional responses to facilitate bacterial growth and survival.Signal Transducer and Activator of Transcription 3(STAT3)is found to be activated by the Salmonella pathogenicity island-1(SPI-I)T3SS effectors SopB,SopE and SopE2.However,the mechanisms of STAT3 activation by S.Typhimurium remain unclear.First,we infected cultured wild-type mouse embryonic fibroblast(MEF)cells with various pathogens including S.Typhimurium,Shigella flexneri,Listeria monocytogenes,and Staphylococcus aureus.A significant portion of modified TRAF6 was observed in S.Typhimurium-infected cells.We found the changes in TRAF6 mobility caused by S.Typhimurium were due to ubiquitination via immunoprecipitation.We constructed a ?sopE2 mutant and a ?sopB/sopE2 double mutant in S.Typhimurium strain LT2.We assure that SopB and SopE2 significantly stimulated the increase in TRAF6 ubiquitination during S.Typhimurium infection.To determine the biological function of TRAF6,we examined the activation of three signaling pathways known to be responsible for the regulation of host transcriptional responses upon S.Typhimurium infection: MAPKs,NF-?B and STAT3.Distinct from MAPKs and NF-?B,TRAF6 deletion substantially diminished STAT3 phosphorylation in Traf6-/-cells compared with the robust induction of STAT3 phosphorylation at Y705 at 8 to 16 hrs post-infection in Traf6+/+ cells.And the time-points of STAT3 pY705 phosphorylation coincided with those of TRAF6 ubiquitination,which is initiated by SPI-1 T3 SS effectors SopB and SopE2,implicating an underlying correlation between TRAF6 and STAT3 phosphorylation at Y705.We mutated Cys70 and Lys124 of TRAF6 to Ala and found that ectopic expression of the C70 A mutant and K124 A mutant in Traf6-/-MEFs not only dramatically impaired the ubiquitination of TRAF6,but simultaneously decreased STAT3 phosphorylation in comparison with wild-type TRAF6 in response to S.Typhimurium infection,confirming the involvement of TRAF6 ubiquitination in STAT3 phosphorylation induced by the SPI-1 T3 SS effectors SopB/SopE2.Our results reveal a novel strategy in which S.Typhimurium T3 SS effectors broaden their functions through the activation of host proteins in a ubiquitination-dependent mechanism to manipulate host cells into a Salmonella-friendly zone.This finding will provide important targets and theoretical basis for looking for potential treatment and improving development of prognosis molecule drug for Salmonella infection.