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. 2020 May 18;12(4):318-321.
doi: 10.1093/jmcb/mjaa003.

Enterohemorrhagic E. coli effector NleL disrupts host NF-κB signaling by targeting multiple host proteins

Xiangpeng Sheng  1   2   3 , Qing You  1   2 , Hongnian Zhu  4 , Qingrun Li  1 , Hong Gao  5 , Haifeng Wang  1 , Chunping You  6 , Qing Meng  4 , Yingjie Nie  7 , Xiangyan Zhang  7 , Ronggui Hu  1
Affiliations

Enterohemorrhagic E. coli effector NleL disrupts host NF-κB signaling by targeting multiple host proteins

Xiangpeng Sheng et al. J Mol Cell Biol. .
No abstract available

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Graphical Abstract
Graphical Abstract
Figure 1
Figure 1
A bacterial effector NleL disrupts host NF-κB pathway by targeting multiple targets. (A) NleL downregulated TNFα-induced NF-κB activation. (B) NleL disrupted p65 translocation from the cytoplasm to the nucleus upon TNFα stimulation. HeLa cells expressing EGFP or EGFP-NleL were subjected to TNFα stimulation. (C) The ability of NleL to inhibit NF-κB pathway activation depended on its intact E3 activity. The NF-κB luciferase activity was measured in cells stimulated by TNFα (10 ng/ml, 6 h). (D) NleL interacted with TRAF2 independent of its E3 activity.(E) NleL interacted with the Zn finger domain (87–264aa) of TRAF2. The cell lysate from HEK293T expressing Flag-TRAF2 or the truncation was subjected to the GST pull-down assay. (F) NleL ubiquitylated TRAF2 in vivo. (G) TRAF2-4KR mutant (K119R/K194R/K201R/K207R) almost abolished NleL-mediated TRAF2 ubiquitylation. (H) NleL suppressed TRAF2 overexpression-induced NF-κB activation. (I) NleL interacted with TRAF proteins. (J) NleL promoted the non-K63-linked ubiquitylation of TRAF5 (left) and TRAF6 (right) in vivo. (K) Luciferase assay in HEK293T cells indicated that NleL attenuated TRAF2-, TRAF5-, or TRAF6-mediated NF-κB activation. (L) NleL formed a complex with several components of the NF-κB pathway. GST-NleL or C753A were incubated with HEK293T cell lysate for pull-down assay. (M) NleL increased the ubiquitylation of IKKα or IKKβ. HA-Ub, Flag-IKK, and His-NleL or C753A were co-expressed in HEK293T cells. Anti-Flag IP was performed in denaturing RIPA buffer. (N) NleL dose-dependently suppressed the activation of NF-κB by IKKα or IKKβ overexpression in HEK293T cells. (O) NleL impaired p65 nuclear translocation in response to TNFα in EHEC infection. HeLa cells stably expressing Jnk1/2 shRNA were infected with EHEC and then subjected to TNFα treatment. Anti-E. coli LPS staining indicated bacteria (green), DAPI staining marked the nucleus (blue), and p65 was shown by anti-p65 antibody in red. (P) The ratio of p65 translocation from cytoplasm to nucleus in each group. At least 10 different views were measured for each group. Statistical significance was determined by Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 10 μm.
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