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

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Enterohemorrhagic Escherichia coli (EHEC) O157:H7, a major diarrheagenic pathogen, can cause bloody diarrhea, hemorrhagic colitis, and >90% of hemolytic uremic syndrome in humans (Mead and Griffin, 1998). Many previous studies have demonstrated that O157:H7 could disrupt host ubiquitin (Ub) system by delivering virulence effectors into host cells with the type III secretion system (T3SS). NleL (also named EspX7) emerged as one of such effectors, whose E3-like activity was first identified in vitro in 2011 (Lin et al., 2011). Our recent work revealed that NleL ubiquitylates human JNK proteins and promotes EHEC-associated A/E lesions (Sheng et al., 2017). However, it remains undetermined whether NleL might mediate other microbe–host interactions that contribute to EHEC infection. Here, we demonstrate that NleL targets several components of the NF-κB pathway to suppress host NF-κB activation.

As the NF-κB pathway is a major target for many bacterial effectors (Neish and Naumann, 2011), we systematically studied the impact of NleL on NF-κB signaling. First, the ectopic expression of NleL in HEK293T cells was shown to dramatically suppress TNFα-mediated p65 phosphorylation (Figure 1A). NleL also attenuated IKK phosphorylation and IκBα degradation (Figure 1A). Second, we found that EGFP-fused NleL, but not EGFP alone, disrupted the nuclear translocation of p65 in response to TNFα, though they have similar localization in HeLa cells at rest (Figure 1B; Supplementary Figure S1A and B). Third, a dual-luciferase NF-κB reporter assay indicated that NleL inhibited TNFα-induced NF-κB luciferase activity (Figure 1C). These results suggest that NleL suppresses NF-κB activation in mammalian cells.

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.

Cys753 is an active site responsible for the E3 ligase activity of NleL and Cys-to-Ala substitution (C753A) abolishes the E3 activity (Lin et al., 2011). We found here that C753A impaired the ability of NleL to suppress NF-κB luciferase activity and p65 translocation into the nucleus (Figure 1C; Supplementary Figure S1C and D), which indicates that the E3 ligase activity of NleL is required to disrupt host NF-κB signaling.

We hypothesized that NleL may target some molecules in the NF-κB pathway. We used recombinant GST-NleL protein to pull down interacted proteins from HEK293T cell lysate. Mass spectrometry identified TRAF2 in the complex pulled down (data not shown). Western blot analysis also revealed that TRAF2 was captured by GST-NleL, but not by GST alone (Supplementary Figure S2A). Consistently, NleL ectopically expressed in HEK293T cells was also co-immunoprecipitated with TRAF2 (Figure 1D; Supplementary Figure S2B). Moreover, there was no significant difference between NleL and C753A mutant to interact with TRAF2 (Figure 1D; Supplementary Figure S2A), indicating that such interaction was independent of the E3 activity. We observed that the Zn finger domain (87–264aa) of TRAF2 was mainly responsible for interacting with NleL (Figure 1E). Altogether, NleL interacts with TRAF2.

We then investigated the ubiquitylation of TRAF2 by NleL. The recombinant TRAF2 protein was ubiquitylated by NleL in vitro (Supplementary Figure S2C). NleL expression in HEK293T cells also readily increased TRAF2 ubiquitylation (Figure 1F). Furthermore, C753A failed to conjugate Ub onto TRAF2 in vitro and in vivo (Figure 1F; Supplementary Figure S2C). Previously, we demonstrated that Ub chains on JNK1 assembled by NleL were predominant in K27, K29, and K33 linkages (Sheng et al., 2017). Similar Ub chain linkages were observed here in NleL-mediated TRAF2 ubiquitylation (Supplementary Figure S2D). Treatment of neither a proteasome inhibitor bortezomib (BTZ) nor a protein synthesis inhibitor cycloheximide (CHX) could regulate TRAF2 protein level, no matter NleL was present or not (Supplementary Figure S2E and F). Thus, NleL-mediated ubiquitylation of TRAF2 did not lead to TRAF2 degradation.

We next mapped the ubiquitylation sites on TRAF2. Mass spectrometry identified 11 potential ubiquitylation sites in TRAF2 (Supplementary Figure S3A). Notably, five sites (K27, K119, K194, K201, and K207) were identified specifically when NleL was present (Supplementary Figure S3A). We generated a series of K-to-R TRAF2 mutants. Four TRAF2 mutations (K119R, K194R, K201R, and K207R) significantly decreased NleL-mediated ubiquitylation (Supplementary Figure S3B–D). Simultaneous mutation in these four residues (4KR) dramatically reduced the ubiquitylation of TRAF2 (Figure 1G), indicating that they are the major ubiquitylation sites. A luciferase assay showed that NleL could suppress TRAF2-induced NF-κB activity, while C753A did not (Figure 1H). Altogether, NleL suppresses the NF-κB pathway by targeting TRAF2.

As there are six well-studied members in TRAF family (TRAF1–6) with similar structures (Xie, 2013; Supplementary Figure S4A), we asked whether NleL might target other TRAF proteins. GST pull-down assays indicated that NleL interacted with all the TRAF proteins, with PABPC1 as an irrelevant and negative control (Figure 1I). Moreover, NleL was capable of ubiquitylating all TRAF proteins except TRAF4 (Figure 1J; Supplementary Figure S4B and C). Particularly, NleL could promote TRAF6 ubiquitylation in vivo and in vitro (Figure 1J; Supplementary Figure S4D). In innate immunity, the K63-linked ubiquitylation of TRAF6 has fundamental functions (Kobayashi et al., 2004). However, NleL could not conjugate the K63-linked Ub chain onto TRAF6 (Figure 1J), suggesting that NleL-mediated ubiquitylation could disrupt the formation of the typical K63-linkage in host cells. Luciferase assays showed that NleL also suppressed TRAF5 or TRAF6 overexpression-induced NF-κB activation (Figure 1K). Thus, NleL suppresses the NF-κB pathway by targeting several TRAF proteins.

IKKs, IκBα, and p65 are key downstream regulators in NF-κB signaling. Intriguingly, either NleL or its C753A mutant was capable of forming a complex with IKKα, IκBα, or p65 (Figure 1L). The interaction between NleL and IKKs was confirmed by the co-immunoprecipitation assay (Supplementary Figure S5A). Deletion of the leucine zipper domain in IKKα compromised IKKα–NleL interaction (Supplementary Figure S5B), in a manner highly reminiscent of the role of zinc finger domain of TRAF2 (Figure 1E). Furthermore, NleL could readily ubiquitylate IKKs (Figure 1M). p65, IκBα, and other important components in the NF-κB pathway, however, could not be ubiquitylated by NleL (Supplementary Figure S5C–F). NleL suppressed IKKs-induced NF-κB activation (Figure 1N) but had no effect on NF-κB activation induced by the p50 or p65 overexpression (Supplementary Figure S5G). Thus, NleL also specifically targets IKKs, but not RIP1, NIK, IκBα, or p50/p65.

We next evaluated the roles of NleL in regulating NF-κB signaling during EHEC infection. Since no good murine models are available for EHEC O157 infection (Mohawk and O'Brien, 2011) and Citrobacter rodentium cannot serve as a suitable surrogate for EHEC O157 to study NleL (Sheng et al., 2017), our infection experiments were only performed in epithelial cells. As NleL enhances EHEC adherence to host cells by targeting JNKs, we used Jnk1/2-knockdown HeLa cells or JNK inhibitor treatment to ensure that each EHEC strain could adhere to mammalian cells with comparable efficiency and then evaluated their abilities to inhibit NF-κB activation. Cells were incubated with a wild-type (WT) EHEC, a T3SS mutant ∆escR, a ∆nleL mutant, or a ∆nleL mutant complemented with NleL (ΔnleL+NleL) or C753A (ΔnleL+C753A). After infection, cells were stimulated with TNFα and next subjected to analyzing p65 translocation (Figure 1O and P). p65 translocation was inhibited more strongly by WT infection than ΔnleL infection. Restoration of NleL, but not C753A, in ΔnleL blocked p65 nuclear translocation. A similar observation occurred in a luciferase assay using EHEC-infected and JNK inhibitor-treated HEK293T cells (Supplementary Figure S6). Therefore, NleL-induced ubiquitylation disrupts host NF-κB signaling in EHEC infection.

In summary, we have identified multiple host targets of NleL. NleL not only ubiquitylates JNK proteins to promote EHEC-induced A/E lesions (Sheng et al., 2017) but also targets TRAF2, TRAF5, TRAF6, IKKα, and IKKβ to disrupt host NF-κB pathway. By unraveling the multitargeting functions of a bacterial effector, our studies may facilitate the comprehensive understanding of the power of bacterial effectors in disrupting host defense. Lastly, as our experiments were mainly performed in the cell culture with ectopic expression of the related entities, future studies are warranted to fully elucidate the pathophysiological relevance of the major findings reported above.

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