Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6) Mediates Ubiquitination-Dependent STAT3 Activation upon Salmonella enterica Serovar Typhimurium Infection

Abstract

Salmonella enterica serovar 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. Here we show that infection of host cells with S. Typhimurium specifically induces the ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6). This TRAF6 ubiquitination is triggered by the Salmonella pathogenicity island 1 (SPI-1) T3SS effectors SopB and SopE2. We also demonstrate that TRAF6 is involved in the SopB/ SopE2-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), a signaling event conducive to the intracellular growth of S. Typhimurium. Specifically, TRAF6 mediates lysine-63 ubiquitination within the Src homology 2 (SH2) domain of STAT3, which is an essential step for STAT3 membrane recruitment and subsequent phosphorylation in response to S. Typhimurium infection. TRAF6 ubiquitination participates in STAT3 phosphorylation rather than serving as only a hallmark of E3 ubiquitin ligase activation. Our results reveal a novel strategy in which S. Typhimurium T3SS effectors broaden their functions through the activation of host proteins in a ubiquitination-dependent manner to manipulate host cells into becoming a Salmonella-friendly zone.KEYWORDS Salmonella enterica serovar Typhimurium, TRAF6, ubiquitination, STAT3, Salmonella effectors I nfection with bacterial pathogens often induces host inflammatory responses (1), which are initiated by the recognition of microbial products, collectively known as pathogen-associated molecular patterns (PAMPs). PAMPs are recognized by Toll-like receptors (TLRs) or cytosolic NOD-like receptors (NLRs) (1, 2), which activate nuclear factor B (NF-B) and mitogen-activated protein kinases (MAPKs) and induce the production of proinflammatory cytokines critical for host defenses (3, 4). To overcome host defenses, some pathogens have developed strategies to dampen the host innate immune response by inactivating MAPKs or NF-B signaling. For instance, the Shigella flexneri phosphothreonine lyase OspF, which is injected into host cells by a type III secretion system (T3SS), inactivates the innate immune response by dephosphorylating MAPKs (5). The Yersinia protein YopJ/YopP, a T3SS effector protein containing both

MicroRNA-127 Promotes Anti-microbial Host Defense through Restricting A20-Mediated De-ubiquitination of STAT3

“…These data, combined with our current findings, demonstrate a coherent link between NF-kB-driven proinflammatory signaling and STAT3mediated anti-microbial pathway, which ensures defensive response at appropriate magnitude and kinetics to avoid unresolved infection (Ji et al, 2019). As an additional support, a recent study demonstrated that TRAF6, the E3 ligase required for NF-kB activation, exerted a boosting role in STAT3 activation through enhancing K63 ubiquitin (Ruan et al, 2017). Interestingly, it has been demonstrated that the deubiquitinase A20 can counteract the activity of TRAF6 through removing K63-ubiquitin from the subsets and feedback regulate vital signal pathways driven by NF-kB and TGF-b1/Smad6 (Shembade et al, 2010;Jung et al, 2013).…”

“…In particular, our data indicate that A20 specifically interacted with STAT3 and inhibited the conjugation of K63-ubiquitin to STAT3, which in turn promoted STAT3 activation in response to staphylococcal infection. Although phosphorylation of STAT3 at Tyr705 or Ser727 has been identified as essential step for STAT3 activity, other post-translation modification and their relevance to STAT3 activation remain to be explored (Cho et al, 2019;Dallavalle et al, 2016;Ruan et al, 2017). We herein prove that de-repression of A20-mediated restraint of STAT3 ubiquitination is required for STAT3 activation and hence anti-bacterial signaling.…”

“…the ubiquitinative modification (Cho et al, 2019;Ruan et al, 2017), we postulated that miR-127 might regulate STAT3 signaling through A20-mediated ubiquitination. To test this, we detected the direct interaction between STAT3 and A20 by Co-IP test.…”

“…Among the most studied polyubiquitinations, Lys48 (K48)-linked ubiquitination enables proteasome-mediated degradation of targeted proteins, whereas K63-linked ubiquitination contributes to protein stabilization and signaling transduction (Heaton et al, 2016). STAT3 has been conjugated with K63ubiquitin by the E3 ligase tumor-necrosis-factor-receptor-associated factor 6 (TRAF6) during the response to Salmonella infection (Ruan et al, 2017). Also, Hectd3-mediated non-degradative ubiquitination constituted the prerequisite for STAT3 activation and contributed to Th17 responses and the resultant neuroinflammatory diseases (Cho et al, 2019).…”

SopB activates the Akt-YAP pathway to promote Salmonella survival within B cells

“…Thus, it is likely that through autocrine mechanisms, IL-10 also contributes to bacterial survival by blocking xenophagy. Additionally, it has been demonstrated that SopB recruits and activates TRAF6, an ubiquitin ligase responsible for STAT3 ubiquitination [58], allowing phosphorylation and activation. STAT3 is a transcription factor necessary for IL-10 production in macrophages; thus, it is possible that SopB-TRAF6 participates in IL-10 gene transcription in Salmonella -infected B cells.…”