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: STAT3; Salmonella effectors; Salmonella enterica serovar Typhimurium; TRAF6; ubiquitination.

FIG 1

S. Typhimurium induces the modification of TRAF6. (A) Induction of TRAF6 modification in MEFs by S. Typhimurium, Shigella flexneri, Listeria monocytogenes, and Staphylococcus aureus. Cultured MEFs were infected with equal amounts of bacteria at an MOI of 10 for 1 h. The infected cells were then chased in the presence of gentamicin and were lysed at the indicated times. The proteins of the infected cells were then separated and subjected to Western blot analysis with rabbit anti-TRAF6, using anti-GAPDH as a loading control. The asterisk indicates the predicted mobility of unmodified TRAF6. (B) Quantified ratio of total TRAF6 to GAPDH (from panel A). Each band was measured with ImageJ, and measurements were normalized to GAPDH values. Values are means (± SD) for three independent experiments. Single and double asterisks indicate statistically significant differences (P < 0.05) in the ratios of TRAF6 to GAPDH within Henle-407 cells and RAW 264.7 cells, respectively, from the values for uninfected cells as determined by Student’s t test.

FIG 2

S. Typhimurium T3SS effector-dependent ubiquitination of TRAF6. (A) TRAF6 is ubiquitinated upon S. Typhimurium infection. Traf6^+/+ MEFs were infected with S. Typhimurium for 8 h. (Left) The presence of ubiquitinated TRAF6 [TRAF6-(Ub)n] in the infected cells was analyzed by immunoprecipitation (IP) with rabbit anti-TRAF6 and Western blotting (7.5% SDS-PAGE gels) (IB) with mouse anti-TRAF6 or mouse anti-ubiquitin. (Right) Whole-cell lysates (WCE) were probed on Western blots from 10% SDS-PAGE gels with rabbit anti-TRAF6, mouse anti-ubiquitin, and anti-GAPDH (as a loading control). (B) Dependence of TRAF6 ubiquitination on the S. Typhimurium effectors SopB and SopE2. Cultured Traf6^+/+ MEFs were infected with wild-type S. Typhimurium or the ΔsopB, ΔsopE2, ΔsopB ΔsopE2, or ΔinvA mutant at an MOI of 10 for 1 h, followed by Western blotting with rabbit anti-TRAF6. (C) Quantification of the ratio of total TRAF6 to GAPDH (from panel B). Values are means (± SD) for three independent experiments. Single or double asterisks indicate statistically significant differences (P < 0.05) between the wild type (WT) and the ΔsopB mutant or between the WT and the ΔsopE2 mutant, respectively, using two-way analysis of variance (ANOVA) with Prism software. (D) Traf6^+/+ MEFs either were not infected (n.i.) or were infected with the indicated S. Typhimurium mutant expressing Yersinia pseudotuberculosis invasin protein at an MOI of 50 or with wild-type S. Typhimurium at an MOI of 10 for 1 h, followed by Western blotting with rabbit anti-TRAF6, using anti-GAPDH as a loading control.

FIG 3

The absence of TRAF6 inhibits the phosphorylation of MAPKs, the activation of NF-κB, and the phosphorylation of STAT3 upon S. Typhimurium infection. (A and B) Infected cells were analyzed by Western blotting with antibodies against MAPKs and IκBα (A) or against STAT3 and the phosphorylated forms of STAT3 (pY705 and pS727) (B). Tubulin was detected as a loading control. (C) Quantification of the fold activation of STAT3-pY705 and STAT3-pS727 (relative to levels in uninfected cells). Values are means (± SD) for three independent experiments. Asterisks indicate statistically significant differences (P < 0.05) from the values for Traf6^+/+ cells as determined by Student's t test.

FIG 4

TRAF6 catalyzes STAT3 ubiquitination upon S. Typhimurium infection. (A) Traf6^+/+ MEFs were first serum starved and then infected with S. Typhimurium at an MOI of 10 for the indicated times. WCEs were collected for immunoprecipitation (IP) with rabbit anti-STAT3, followed by Western blotting (IB) with mouse anti-STAT3 and mouse anti-ubiquitin. WCEs were probed with rabbit anti-STAT3 and rabbit anti-tubulin (as a loading control). (B) Importance of the TRAF6 C70 residue for S. Typhimurium-induced STAT3 ubiquitination and phosphorylation. Traf6^−/− MEFs were transfected with either mock plasmids (Vector), pcDNA4-FLAG-TRAF6 (WT), or pcDNA4-FLAG-TRAF6 C70A (C70A) for 24 h before infection with S. Typhimurium for 8 h. STAT3 was immunoprecipitated as described for panel A, and the phosphorylation of STAT3 was detected as for Fig. 3B.

FIG 5

Mutation of lysines within the SH2 domain abolishes STAT3 ubiquitination. (A) Ubiquitination and phosphorylation of FLAG-STAT3 or FLAG-STAT3 ΔSH2 when cotransfected with HA-TRAF6 into HEK293T cells for 24 h before infection with S. Typhimurium for 8 h. FLAG-STAT3 or FLAG-STAT3 ΔSH2 was pulled down using anti-FLAG M2 magnetic beads and was then analyzed with rabbit anti-STAT3 and rabbit anti-STAT3-pY705. WCEs were probed with mouse anti-FLAG and rabbit anti-tubulin (as a loading control). (B) Diagram indicating the locations of lysine residues within the SH2 domain of STAT3. (C) Effects of mutations in the STAT3 SH2 domain lysine residues on STAT3 ubiquitination and phosphorylation. FLAG-STAT3 or FLAG-STAT3 K(1-6)A was cotransfected with HA-TRAF6 into HEK293T cells for 24 h before infection with S. Typhimurium for the indicated times. FLAG-STAT3 was pulled down and detected as described for panel A. (D) Effects of mutations in the STAT3 SH2 domain lysine residues on STAT3 membrane localization and phosphorylation. FLAG-STAT3 or FLAG-STAT3 K(1-6)A was cotransfected with HA-TRAF6 into HEK293T cells for 24 h before infection with S. Typhimurium for 8 h. WCE and plasma membrane (Mem) fractions were analyzed by Western blotting with the indicated antibodies.

FIG 6

TRAF6 ubiquitination is important for the induction of STAT3 phosphorylation by S. Typhimurium. (A) Importance of the TRAF6 K124 residue in STAT3 phosphorylation. Traf6^−/− MEFs were transfected with mock plasmids, pcDNA4-FLAG-TRAF6, pcDNA4-FLAG-TRAF6 C70A, or pcDNA4-FLAG-TRAF6 K124A for 24 h before infection with S. Typhimurium for 8 h. WCEs were probed with mouse anti-FLAG, rabbit anti-pSTAT3 (Y705), rabbit anti-STAT3, or anti-GAPDH (as a loading control). (B) Quantification of the fold activation of STAT3-pY705 (relative to that for Traf6^−/− MEFs expressing a mock plasmid). Values are means (± SD) for three independent experiments. Asterisks indicate statistically significant (P < 0.05) differences from the values for Traf6^−/− MEFs expressing wild-type TRAF6 as determined by Student's t test. (C) Schematic representation. SPI-1 T3SS effectors SopB and SopE2 initiate the ubiquitination of TRAF6 via the activation of RacI, Cdc42, and RhoG. TRAF6 catalyzes STAT3 ubiquitination, which is essential for STAT3 membrane recruitment and subsequent phosphorylation. TRAF6 ubiquitination not only acts as a hallmark of activation of ubiquitin ligase activity but also is involved in TRAF6-catalyzed STAT3 ubiquitination. STAT3 phosphorylation modulates host gene expression to promote intracellular bacterial replication.