Identification of TRAF6 as a ubiquitin ligase engaged in the ubiquitination of SopB, a virulence effector protein secreted by Salmonella typhimurium.
The phosphoinositide phosphatase SopB is one of the effectors injected by Salmonella typhimurium (S.typhimurium) that diversifies its function through a ubiquitin-dependent differential localization. However, it is unclear which E3 ubiquitin ligase is responsible for ubiquitination of SopB. Based on the E1-E2-E3 trio of enzymes responsible for the ubiquitin activation and translocation to substrate proteins, we constructed an in vitro assay of SopB ubiquitination. Using this assay, we purified an E3 ubiquitin ligase, TRAF6, from the Henle-407 S100 extraction that may be responsible for the ubiquitination of SopB. To investigate the functional correlation of TRAF6, we showed that recombinant TRAF6 specifically ubiquitinates SopB in a dose-dependent manner in vitro. Upon infection, the ubiquitination of SopB was absolutely blocked by TRAF6 deletion, as shown in Traf6(-/-) mouse embryonic fibroblasts (MEFs) compared with Traf6(+/+) MEFs. However, the ectopic expression of TRAF6 in Traf6(-/-) MEFs rescued the two species of ubiquitin-conjugated SopB, which strengthens the role of TRAF6 in SopB ubiquitination. The analysis of E2 revealed that UbcH5c and not other E2 conjugating enzymes are required for TRAF6-mediated SopB ubiquitination both in vitro and in vivo. In summary, these results suggest the relevance of UbcH5c/TRAF6 in SopB during S.typhimurium infection and thereby imply that S.typhimurium has evolved a mechanism of utilizing the host's E3 ubiquitin ligase to modify and modulate the function of its effector protein in order to ensure pathogen and host cell survival.
Tianjin Key Laboratory of Food Science and Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China. Electronic address: ruanhaihua@tjcu.edu.cn.
Hai-Hua
HH
Ruan
Tianjin Key Laboratory of Food Science and Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China.
Ye
Y
Li
Tianjin Key Laboratory of Food Science and Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China.
Xi-Xuan
XX
Zhang
Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Molecular Nuclear Medicine, Tianjin 300192, China.
Qiang
Q
Liu
Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University Cancer Hospital, Tianjin 300071, China.
He
H
Ren
Tianjin Key Laboratory of Food Science and Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China.
Kun-Sheng
KS
Zhang
Tianjin Key Laboratory of Food Science and Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China. Electronic address: zhaohui@tjcu.edu.cn.
Hui
H
Zhao
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D005347:Fibroblasts; D051379:Mice; D012486:Salmonella typhimurium / Q000235:genetics; D048029:TNF Receptor-Associated Factor 6 / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins; D048029:TNF Receptor-Associated Factor 6; C585419:UBE2D3 protein, mouse; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C489224:SopB protein, Salmonella
Effector; Salmonella typhimurium; SopB; TRAF6; Ubiquitin ligase; Ubiquitination