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.