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. 2007 Oct 31;26(21):4457-66.
doi: 10.1038/sj.emboj.7601867. Epub 2007 Oct 4.

Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species

Amrita Kumar  1 , Huixia Wu, Lauren S Collier-Hyams, Jason M Hansen, Tengguo Li, Kosj Yamoah, Zhen-Qiang Pan, Dean P Jones, Andrew S Neish
Affiliations

Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species

Amrita Kumar et al. EMBO J. .

Abstract

The resident prokaryotic microflora of the mammalian intestine influences diverse homeostatic functions of the gut, including regulation of cellular growth and immune responses; however, it is unknown how commensal prokaryotic organisms mechanistically influence eukaryotic signaling networks VSports手机版. We have shown that bacterial coculture with intestinal epithelial cells modulates ubiquitin-mediated degradation of important signaling intermediates, including beta-catenin and the NF-kappaB inhibitor IkappaB-alpha. Ubiquitination of these proteins as well as others is catalyzed by the SCF(betaTrCP) ubiquitin ligase, which itself requires regulated modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Here we show that epithelia contacted by enteric commensal bacteria in vitro and in vivo rapidly generate reactive oxygen species (ROS). Bacterially induced ROS causes oxidative inactivation of the catalytic cysteine residue of Ubc12, the NEDD8-conjugating enzyme, resulting in complete but transient loss of cullin-1 neddylation and consequent effects on NF-kappaB and beta-catenin signaling. Our results demonstrate that commensal bacteria directly modulate a critical control point of the ubiquitin-proteasome system, and suggest how enteric commensal bacterial flora influences the regulatory pathways of the mammalian intestinal epithelia. .

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Figures

Figure 1
Figure 1
Commensal bacteria induce generation of epithelial ROS and cause oxidative stress. (A, B) Commensal bacteria cause oxidation of Trx1 pools. Caco2 cells were colonized with different commensal bacterial strains (L. rhamnosus (open bars), B. thetaiotaomicron (bars with diagonals), E. coli (dotted bars)) for 15 min as shown in panel A or with Lactobacillus over a time course as shown in panel B. Cells treated with PBS served as controls (filled bars). The graphs show the Eh for Trx1, and data are represented as means±s.e. All values denote a statistically significant difference from control (P<0.05). (C, D) Redox analysis of GSH/GSSG following colonization of IEC-6 cells with different commensal bacterial strains (L. rhamnosus (open bars), B. thetaiotaomicron ( bars with diagonals), E. coli (dotted bars)) for 15 min as shown in panel C or with Lactobacillus over a period of time as shown in panel D. Cells treated with PBS served as control (open bars). Graphs show the Eh for GSH/GSSG. Data are represented as means±s.e. All values denote a statistically significant difference from control (P<0.05). (E) Bacteria stimulate rapid epithelial ROS generation. IEC-6 cells were colonized with Lactobacillus, washed, and incubated with 5 μM of CM-H2DCF-DA. Fluorescence was measured by confocal laser scanning microscopy (Zeiss). (F) Kinetics of bacterially induced ROS generation in epithelial cells. ROS production was measured in trypsinized HeLa cells incubated with Lactobacillus by luminol assay. Peak relative luminescence is shown. Data represent the mean of three independent assays and are expressed in units of luminol chemiluminescence (LmCL).
Figure 2
Figure 2
In vivo changes in intracellular redox in mouse intestinal epithelial cells contacted by commensal bacteria. (A) Lactobacillus-induced Trx1 oxidation in epithelial cells as analyzed by redox Western analysis. The graph shows the Eh values for Trx1 in proximal jejunum 30 min after treatment with a bolus of bacteria (open bars) as compared with untreated controls (filled bars). Data are represented as means±s.e. Asterisks denote a statistically significant difference from control (P<0.05). (B) Lactobacillus-induced changes in the redox pools of GSH/GSSG. The graph shows the Eh values for GSH/GSSG in the jejunum 30 min after treatment with a bolus of bacteria (open bars) as compared with untreated controls (filled bars). Data are represented as means±s.e. The asterisks denote a statistically significant difference from control (P<0.05). (C) In situ detection of superoxide in mouse colon treated with Lactobacillus. After 30 min of infusion of Lactobacillus, mouse colon was stained with DHE and imaged as described. Under identical tissue processing and imaging conditions, nuclear fluorescence in mouse colon treated with bacteria (top panels) is markedly increased compared with colon from untreated control mouse (bottom panels).
Figure 3
Figure 3
Loss of cullin-1 neddylation by bacterially mediated ROS generation. (A) Bacteria cause a time-dependent loss of Cul-1 neddylation that is reversible. Cul-1 is detected in whole-cell protein extracted from HeLa cells incubated with Lactobacillus and immunoblotted with Cul-1-specific antiserum. (B) Bacteria cause a time-dependent loss of Cul-1 neddylation in vivo. Western blot analysis with Cul-1-specific antisera of proteins extracted from mouse intestinal epithelial cells treated with bacteria via an ileal loop. (C) H2O2 causes time- and dose-dependent loss of Cul-1 neddylation. HeLa cells treated with H2O2 were analyzed as in panel A. (D) DPI and NAC prevent bacterially mediated loss of Cul-1 neddylation. HeLa cells pretreated with NAC or DPI for 30 min and colonized with Lactobacillus for 30 min were analyzed as in panel A. (E) Un-neddylated Cul-1 associates with IκB-α and does not support ubiquitination. Immunoprecipitation of IκB-Flag with anti-Flag agarose and immunoblots with IκB- and Cul-1-specific antisera of lysates from transfected HeLa cells challenged with TNF-α for 30 min or colonized by bacteria in the presence of the proteasome inhibitor MG-262. Note that the hyperneddylation of IκB-associated Cul-1 in TNF-α-treated cells is consistent with Cul-1 being in an active state to support ubiquitination of IκB and allow subsequent NF-κB activation.
Figure 4
Figure 4
Oxidant modulation of E3-SCFβTrCP mediated signaling. (A) H2O2 prevents TNF-α-induced p65 nuclear translocation. Immunofluorescent staining of p65 in HeLa cells preincubated with 1 mM H2O2 for 45 min and treated with TNF-α for 30 min. (B) H2O2 inhibits activity of a NF-κB-luciferase reporter. Lysates from HeLa cells transfected with a NF-κB-luciferase reporter plasmid, preincubated with H2O2 for 45 min, and treated in the presence of H2O2 with TNF-α for 5 h were assayed for luciferase activity. Data represent the mean of three independent assays and are shown as % TNF-α-induced NF-κB-Luc. (C) H2O2 prevents ubiquitination of β-catenin. Western blot analysis with anti-β-catenin and anti-phospho-β-catenin Abs of whole-cell protein extracted from epithelial cells pretreated 1 mM H2O2 for 1 h, washed, and treated with MG-262 (500 nM) for the indicated time. (D) H2O2 causes increased accumulation of β-catenin. Western blot analysis with anti-β-catenin and anti-β-actin Abs of whole-cell protein extracted from HeLa cells treated with HBSS±1 mM H2O2 for the indicated times. (E) H2O2 induces nuclear translocation of β-catenin. Confocal microscopy of HeLa cells treated with HBSS±1 mM H2O2 for the indicated times, and stained with the anti-β-catenin antibody (green) and nuclei counterstained with To-Pro-3 iodide (TP-3, blue). Merged images display sites of co-labeling.
Figure 5
Figure 5
Bacterially induced oxidant stress causes inactivation of the neddylation machinery. (A) Inhibition of Cul-1 neddylation in vitro by H2O2. In vitro neddylation was carried out in the presence of H2O2 using purified Ubc12, a complex containing Roc1 and the C-terminal portion (residues 324–776) of Cul-1, and 32P-NEDD8. (B) H2O2 inhibits the formation of the NEDD8∼Ubc12 thioester bond and leads to the formation of a DTT-sensitive HMW Ubc12 species. Western blot analysis with RH- and NEDD8-specific antisera of whole-cell protein extracted from HeLa cells transfected with WT or mutant (C111A or C111S) RH-Ubc12, treated with H2O2 for 1 h at the indicated dose, and lysed in SDS lysis buffer without (−DTT) or with (+DTT) reducing agents. The star marks the 30-kDa H2O2-sensitive NEDD8∼Ubc12 thioester form. (C) H2O2 induces the loss of NEDD8∼Ubc12 thioester bond at low doses. HeLa cells transfected with RH-Ubc12 (WT, C111A or C111S) were treated with increasing H2O2 concentrations for 1 h, lysed in SDS lysis buffer without DTT and immunoblotted with anti-RH antibody. (D) In vitro treatment of purified Ubc12 with H2O2 results in the oxidation of Ubc12. Purified Ubc12 (WT) and Ubc12 (C111A) were treated with H2O2 (0.1–1 mM) and labeled with the thiol-sensitive dyes BIAM or F5M. Samples and control were separated on a 15% PAGE and labeled protein was detected as described. A parallel gel was immediately probed with anti-RH antibody to identify specific protein band. (E) Bacteria or H2O2 inhibit the formation of the NEDD8∼Ubc12 thioester bond of endogenous Ubc12 in vitro. Western blot analysis with Ubc12- and Cul-1-specific antisera of whole-cell protein extracted from HeLa cells treated with 1 mM H2O2 or Lactobacillus and lysed in SDS lysis buffer without (−DTT, top panels) or with (+DTT, bottom panels) reducing agents. The stars mark the 30-kDa H2O2- and colonization-sensitive NEDD8∼Ubc12 thioester forms. (F) Bacteria inhibit the formation of the NEDD8∼Ubc12 thioester in vivo. Western blot analysis with Ubc12-specific antisera of proteins extracted from mouse intestinal epithelial cells treated with bacteria via an ileal loop.
Figure 6
Figure 6
Model of bacterially induced ROS generation on signaling pathways. Summary diagram of bacterial inhibition of SCF signaling. Bacteria elicit ROS generation in epithelial cells. ROS inactivate the Ubc12 enzyme, preventing the neddylation of cullin-1. Un-neddylated cullin in the E3-SCFβ−TrCP complex renders it unable to carry out ubiquitination and is thus inactive.
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