Salmonella Effector AvrA Regulation of Colonic Epithelial Cell Inflammation by Deubiquitination
2007; Elsevier BV; Volume: 171; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.070220
ISSN1525-2191
AutoresZhongde Ye, Elaine O. Petrof, David L. Boone, Erika C. Claud, Jun Sun,
Tópico(s)Amoebic Infections and Treatments
ResumoAvrA is a newly described bacterial effector existing in Salmonella. Here, we test the hypothesis that AvrA is a deubiquitinase that removes ubiquitin from two inhibitors of the nuclear factor-κB (NF-κB) pathway, IκBα and β-catenin, thereby inhibiting the inflammatory responses of the host. The role of AvrA was assessed in intestinal epithelial cell models and in mouse models infected with AvrA-deficient and -sufficient Salmonella strains. We also purified AvrA and AvrA mutant proteins and characterized their deubiquitinase activity in a cell-free system. We investigated target gene and inflammatory cytokine expression, as well as effects on epithelial cell proliferation and apoptosis induced by AvrA-deficient and -sufficient bacterial strains in vivo. Our results show that AvrA blocks degradation of IκBα and β-catenin in epithelial cells. AvrA deubiquitinates IκBα, which blocks its degradation and leads to the inhibition of NF-κB activation. Target genes of the NF-κB pathway, such as interleukin-6, were correspondingly down-regulated during bacterial infection with Salmonella expressing AvrA. AvrA also deubiquitinates and thus blocks degradation of β-catenin. Target genes of the β-catenin pathway, such as c-myc and cyclinD1, were correspondingly up-regulated with AvrA expression. Increased β-catenin further negatively regulates the NF-κB pathway. Our findings suggest an important role for AvrA in regulating host inflammatory responses through NF-κB and β-catenin pathways. AvrA is a newly described bacterial effector existing in Salmonella. Here, we test the hypothesis that AvrA is a deubiquitinase that removes ubiquitin from two inhibitors of the nuclear factor-κB (NF-κB) pathway, IκBα and β-catenin, thereby inhibiting the inflammatory responses of the host. The role of AvrA was assessed in intestinal epithelial cell models and in mouse models infected with AvrA-deficient and -sufficient Salmonella strains. We also purified AvrA and AvrA mutant proteins and characterized their deubiquitinase activity in a cell-free system. We investigated target gene and inflammatory cytokine expression, as well as effects on epithelial cell proliferation and apoptosis induced by AvrA-deficient and -sufficient bacterial strains in vivo. Our results show that AvrA blocks degradation of IκBα and β-catenin in epithelial cells. AvrA deubiquitinates IκBα, which blocks its degradation and leads to the inhibition of NF-κB activation. Target genes of the NF-κB pathway, such as interleukin-6, were correspondingly down-regulated during bacterial infection with Salmonella expressing AvrA. AvrA also deubiquitinates and thus blocks degradation of β-catenin. Target genes of the β-catenin pathway, such as c-myc and cyclinD1, were correspondingly up-regulated with AvrA expression. Increased β-catenin further negatively regulates the NF-κB pathway. Our findings suggest an important role for AvrA in regulating host inflammatory responses through NF-κB and β-catenin pathways. Every year, approximately 40,000 cases of salmonellosis are reported in the United States. Most persons infected with Salmonella develop diarrhea, fever, and abdominal cramps 12 to 72 hours after infection. AvrA is a Salmonella effector translocated into host cells by a type 3 secretion system. Recently studies showed that the AvrA gene is present in 80% of Salmonella enterica serovar.1Streckel W Wolff AC Prager R Tietze E Tschèape H Expression profiles of effector proteins SopB, SopD1, SopE1, and AvrA differ with systemic, enteric, and epidemic strains of Salmonella enterica.Mol Nutr Food Res. 2004; 48: 496-503Crossref PubMed Scopus (35) Google Scholar Although its exact function is not entirely clear, AvrA belongs to the family of cysteine proteases regulating diverse bacterial-host interactions.2Orth K Xu Z Mudgett MB Bao ZQ Palmer LE Bliska JB Mangel WF Staskawicz B Dixon JE Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease.Science. 2000; 290: 1594-1597Crossref PubMed Scopus (436) Google Scholar Other family members related to AvrA include the adenovirus protease AVP, Yersinia virulence factor YopJ (Yersinia outer protein J), and the Xanthomonas campestris pv. vesicatoria protein AvrBsT. Wild-type Salmonella activates the nuclear factor-κB (NF-κB) pathway, whereas nonvirulent Salmonella strains, such as PhoPc, attenuate the host innate immune response by preventing the activation of the NF-κB pathway.3Neish AS Gewirtz AT Zeng H Young AN Hobert ME Karmali V Rao AS Madara JL Prokaryotic regulation of epithelial responses by inhibition of IκB-α ubiquitination.Science. 2000; 289: 1560-1563Crossref PubMed Scopus (747) Google Scholar The AvrA protein from nonpathogenic Salmonella typhimurium has been shown to inhibit activation of NF-κB in cultured human epithelial cells.4Collier-Hyams LS Zeng H Sun J Tomlinson AD Bao ZQ Chen H Madara JL Orth K Neish AS Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-κB pathway.J Immunol. 2002; 169: 2846-2850PubMed Google Scholar Mutation of the conserved catalytic cysteine in AvrA abolishes its ability to inhibit the proinflammatory NF-κB pathways that are activated within infected cells.4Collier-Hyams LS Zeng H Sun J Tomlinson AD Bao ZQ Chen H Madara JL Orth K Neish AS Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-κB pathway.J Immunol. 2002; 169: 2846-2850PubMed Google Scholar However, the mechanism of AvrA regulation of NF-κB pathways is not completely known. NF-κB controls the expression of many cytokines and chemokines involved in inflammation and immune responses. NF-κB activity is inhibited by the inhibitor of κB (IκBα). IκBα binds to NF-κB to mask the nuclear localization signal so that the NF-κB dimer (p50 and p65) is retained in the cytoplasm. Phosphorylation of IκBα by IκB kinase (IKK) leads to the ubiquitination and degradation of IκBα, resulting in nuclear translocation and activation of NF-κB.5Bonizzi G Karin M The two NF-κB activation pathways and their role in innate and adaptive immunity.Trends Immunol. 2004; 25: 280-288Abstract Full Text Full Text PDF PubMed Scopus (2067) Google Scholar β-Catenin is another protein that has been shown to be a negative regulator of the proinflammatory NF-κB pathway in epithelial cells.6Deng J Miller SA Wang HY Xia W Wen Y Zhou BP Li Y Lin SY Hung MC β-Catenin interacts with and inhibits NF-κB in human colon and breast cancer.Cancer Cell. 2002; 2: 323-334Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 7Deng J Xia W Miller SA Wen Y Wang HY Hung MC Crossregulation of NF-κB by the APC/GSK-3β/β-catenin pathway.Mol Carcinog. 2004; 39: 139-146Crossref PubMed Scopus (86) Google Scholar, 8Sun J Hobert ME Duan Y Rao AS He TC Chang EB Madara JL Crosstalk between NF-κB and beta-catenin pathways in bacterial-colonized intestinal epithelial cells.Am J Physiol. 2005; 289: G129-G137Google Scholar, 9Duan Y Liao AP Kuppireddi S Ye Z Ciancio MJ Sun J β-Catenin activity negatively regulates bacteria-induced inflammation.Lab Invest. 2007; 87: 613-624Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar This function is in addition to its roles in embryonic development and neoplasia such as colon cancer10Morin PJ β-Catenin signaling and cancer.Bioessays. 1999; 21: 1021-1030Crossref PubMed Scopus (813) Google Scholar via enhancement of epithelial cell proliferation. AvrA expression in Salmonella is able to stabilize β-catenin by inhibition of ubiquitination.11Sun J Hobert ME Rao AS Neish AS Madara JL Bacterial activation of β-catenin signaling in human epithelia.Am J Physiol. 2004; 287: G220-G227Google Scholar Polyubiquitination targets proteins for recognition and processing by the 26S proteasome, which degrades the ubiquitinated proteins and recycles ubiquitin. The attachment of ubiquitin to the target proteins requires a series of ATP-dependent enzymatic steps by E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligating) enzymes. Interestingly, both IκBα and β-catenin are targeted for ubiquitination by a similar E3 ligase complex.12Winston JT Strack P Beer-Romero P Chu CY Elledge SJ Harper JW The SCFβ-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IκBα and β-catenin and stimulates IκBα ubiquitination in vitro.Genes Dev. 1999; 13: 270-283Crossref PubMed Scopus (808) Google Scholar The role of AvrA in an in vivo system and the mechanism by which AvrA inhibits NF-κB or activates β-catenin signaling remain unknown. It has been suggested that injection of Salmonella AvrA stabilizes both IκBα and β-catenin.4Collier-Hyams LS Zeng H Sun J Tomlinson AD Bao ZQ Chen H Madara JL Orth K Neish AS Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-κB pathway.J Immunol. 2002; 169: 2846-2850PubMed Google Scholar, 11Sun J Hobert ME Rao AS Neish AS Madara JL Bacterial activation of β-catenin signaling in human epithelia.Am J Physiol. 2004; 287: G220-G227Google Scholar In this study, we test our hypothesis that AvrA is a deubiquitinase, which removes the ubiquitin from both IκBα and β-catenin. We have tested the role of AvrA in nonvirulent PhoPc with sufficient or deficient AvrA expression.4Collier-Hyams LS Zeng H Sun J Tomlinson AD Bao ZQ Chen H Madara JL Orth K Neish AS Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-κB pathway.J Immunol. 2002; 169: 2846-2850PubMed Google Scholar, 11Sun J Hobert ME Rao AS Neish AS Madara JL Bacterial activation of β-catenin signaling in human epithelia.Am J Physiol. 2004; 287: G220-G227Google Scholar PhoPc is a PhoP-PhoQ constitutive mutation of a wild-type S. typhimurium strain that increases the expression of PhoP-activated genes, represses the synthesis of approximately 20 proteins encoded by the PhoP-repressed genes, and attenuates virulence.13Miller SI Mekalanos JJ Constitutive expression of the phoP regulon attenuates Salmonella virulence and survival within macrophages.J Bacteriol. 1990; 172: 2485-2490Crossref PubMed Scopus (312) Google Scholar We demonstrate that Salmonella with AvrA overexpression stabilizes both IκBα and β-catenin in mouse models. In addition, we purified AvrA and AvrA mutant proteins and characterized their deubiquitinase activity in a cell-free system. While examining changes in target gene expression, we also investigated inflammatory cytokine expression, as well as effects on epithelial cell proliferation and apoptosis induced by AvrA-deficient and -sufficient bacterial strains. Our findings strongly suggest that AvrA is a deubiquitinase that regulates both the NF-κB and β-catenin signaling pathways in intestinal inflammation. Bacterial strains S. typhimurium mutant PhoPc, PhoPc AvrA−, and PhoPc AvrA−/AvrA+ were provided by Dr. Andrew Neish and Dr. Lauren Collier-Hyams from Emory University (Atlanta, GA). Bacterial growth conditions were as follows: nonagitated microaerophilic bacterial cultures were prepared by inoculation of 10 ml of Luria-Bertani broth with 0.01 ml of a stationary phase culture, followed by overnight incubation (∼18 hours) at 37°C, as previously described.14McCormick BA Colgan SP Delp-Archer C Miller SI Madara JL Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils.J Cell Biol. 1993; 123: 895-907Crossref PubMed Scopus (382) Google Scholar Bacterial overnight cultures were concentrated 33-fold in Hanks' balanced salt solution (HBSS) supplemented with 10 mmol/L HEPES, pH 7.4. Animal experiments were performed using specific-pathogen-free female C57BL/6 mice (Taconic Farms, Germantown, NY) that were 6 to 7 weeks old. Water and food were withdrawn 4 hours before oral gavage with 7.5 mg/mouse of streptomycin (75 μl of sterile solution or 75 μl of sterile water as control). Afterward, animals were supplied with water and food. Twenty hours after streptomycin treatment, water and food were withdrawn again for 4 hours before the mice were infected with 1 × 107 colony-forming units of S. typhimurium (50-μl suspension in HBSS) or treated with sterile HBSS (control) by oral gavage as previously described.9Duan Y Liao AP Kuppireddi S Ye Z Ciancio MJ Sun J β-Catenin activity negatively regulates bacteria-induced inflammation.Lab Invest. 2007; 87: 613-624Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 14McCormick BA Colgan SP Delp-Archer C Miller SI Madara JL Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils.J Cell Biol. 1993; 123: 895-907Crossref PubMed Scopus (382) Google Scholar At 6, 18, or 24 hours after infection, mice were sacrificed, and tissue samples from the intestinal tracts were removed for analysis. The histological studies, such as studies with hematoxylin and eosin staining, were performed on intestinal segments isolated from mice treated with AvrA-sufficient or -deficient bacterial strains. Cells were rinsed twice in ice-cold HBSS and lysed in ice-cold immunoprecipitation buffer [1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris, pH 7.4, 1 mmol/L ethylenediamine tetraacetic acid (EDTA), 1 mmol/L ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, pH 8.0, 0.2 mmol/L sodium orthovanadate, and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland)]. Samples were prepared as previously described.8Sun J Hobert ME Duan Y Rao AS He TC Chang EB Madara JL Crosstalk between NF-κB and beta-catenin pathways in bacterial-colonized intestinal epithelial cells.Am J Physiol. 2005; 289: G129-G137Google Scholar Blots were probed with anti-β-catenin antibody (BD Biosciences Transduction Laboratories, Lexington, KY). Mouse epithelial cells were scraped and lysed in lysis buffer (1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris, pH 7.4, 1 mmol/L EDTA, 1 mmol/L ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, pH 8.0, 0.2 mmol/L sodium orthovanadate, and protease inhibitor cocktail) and protein concentration measured. Cultured cells were rinsed twice in ice-cold HBSS, lysed in protein-loading buffer [50 mmol/L Tris, pH 6.8, 100 mmol/L dithiothreitol, 2% sodium dodecyl sulfate (SDS), 0.1% bromphenol blue, and 10% glycerol], and sonicated. Equal amounts of proteins or equal volumes of total cultured cell lysates were separated by SDS- polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with anti-β-catenin (BD Biosciences Transduction Laboratories), anti-IκBα, anti-c-myc, anti-cyclinD1 (Santa Cruz Biotechnology, Santa Cruz, CA), or β-actin (Sigma, St. Louis, MO) primary antibodies (1:500 to 1:1000 dilution) and visualized by enhanced chemiluminescence. Chemiluminescent signals were collected and scanned from Hyperfilm ECL (GE Healthcare, Little Chalfont, Buckinghamshire, UK) with a Scanjet 7400c backlit flatbed scanner (Hewlett-Packard Co., Palo Alto, CA). For figures, the contrast of images was adjusted, arranged, and labeled in Adobe Photoshop and Adobe Illustrator (Adobe Systems Incorporated, San Jose, CA). Bands were quantified using NIH Image software (Bethesda, MD). The digital images are representative of the original data. Total mRNA was extracted from scraping mouse colonic epithelial cells using TRIzol reagent (Invitrogen, Carlsbad, CA) and reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) and then subjected to real-time quantitative polymerase chain reaction (PCR) (SYBR Green PCR kit; Bio-Rad) with primers for mouse interleukin (IL)-6 (forward 5′-CAATTCCAGAAACCGCTATGA- 3′, reverse 5′-ACCACAGTGAGGAATGTCCA-3′), c-Myc (forward 5′-TGAGCCCCTAGTGCTGCAT-3′, reverse 5′-AGCCCGACTCCGACCTCTT-3′), and cyclin D1 (forward 5′-AGGTAATTTGCACACCTCTG-3′, reverse 5′-ACAAAGCAATGAGAATCTGG-3′). All expression levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase levels of the same sample, using forward (5-CTTCACCACCATGGAGAAGGC-3′) and reverse (5′-GGCATGGACTGTGGTCATGAG-3′) primers. Percent expression was calculated as the ratio of the normalized value of each sample to that of the corresponding untreated control cells. All real-time PCRs were performed in triplicate. All PCR primers were designed using Lasergene software (DNAStar, Inc., Madison, WI). Before terminating the animal experiment, mouse blood samples were collected by cardiac puncture and placed in tubes containing EDTA (10 mg/ml). Serum was obtained, and mouse IL-6 was measured using the TiterZyme Enzyme Immunometric Assay kit (Assay Designs, Inc., Ann Arbor, MI) according to the manufacturer's instructions. The avrA gene is from wild-type S. typhimurium strain SL3201. Sequence analysis revealed that the avrA allele used in our study is identical to the allele from S. typhimurium LT2 (GenBank accession no. AE008830). Salmonella full-length gene AvrA and one-point mutant C186A were cloned into N-terminal glutathione S-transferase (GST)-fused Vector pEGX-4T2 (Invitrogen) and transformed into Escherichia coli strain BL21(DE3). Bacteria were inoculated in 500 ml of Luria-Bertani broth media with 100 μg/ml ampicillin; once the optical density reached 0.6, bacteria were induced with 0.1 to 1 mmol/L final concentration isopropyl β-d-thiogalactoside for another 3 hours. BL21 cells were spun down, washed with phophate-buffered saline (PBS) twice, and lysed in 5 ml of lysis buffer (50 mmol/L Tris, pH 7.7, 100 mmol/L NaCl, and 0.2 mmol/L EDTA) with a protease inhibitor tablet plus 200 μl of lysozyme stock (20 μg/ml). Samples were mixed well and followed by sonication for 5 seconds three times. Samples were spun down at high speed for 30 minutes at 4°C. Affinity purification was performed by using a glutathione-Sepharose resin (Amersham Bioscience, Piscataway, NJ) and further purified by ion exchange. Human embryonic kidney 293 cells and epithelial HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin-streptomycin, and l-glutamine. Human colonic epithelial HCT116 cells were cultured in McCoy's 5A medium supplemented with 10% (v/v) fetal bovine serum as previously described.8Sun J Hobert ME Duan Y Rao AS He TC Chang EB Madara JL Crosstalk between NF-κB and beta-catenin pathways in bacterial-colonized intestinal epithelial cells.Am J Physiol. 2005; 289: G129-G137Google Scholar The rat small intestinal IEC-18 cell line was grown in Dulbecco's modified Eagle's medium (high-glucose, 4.5 g/L) containing 5% (v/v) fetal bovine serum, 0.1 U/ml insulin, 50 μg/ml streptomycin, and 50 U/ml penicillin. Cells were transiently cotransfected with 1 μg of pGL3-OT [T-cell factor (TCF)-responsive reporter with wild-type TCF binding site] or pGL3-OF (mutant TCF binding site) using Lipofectin reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). pRL-TK vector (Promega, Madison, WI) was used as an internal control reporter. Cells were colonized with equal numbers of bacteria for 30 minutes, washed, and incubated in Dulbecco's modified Eagle's medium for 6 hours. Luciferase activity was monitored using the dual luciferase assay system (Promega). To generate the ubiquitinated IκBα substrates for the reaction, we transfected T7-IκBα into human embryonic kidney 293T cells with Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Plasmids Flag-Fwd1, flag-IKK1, HA-Ub, and T7-IκBα were gifts from Dr. B.E. Wadzinski (Vanderbilt University Medical Center, Nashville, TN15Strayhorn WD Wadzinski BE A novel in vitro assay for deubiquitination of IκBα.Arch Biochem Biophys. 2002; 400: 76-84Crossref PubMed Scopus (24) Google Scholar). All transfections were performed in 60-mm dishes with 8-μg total DNA that included 1 μg of HA-ubiquitin, 1 μg of pcDNA3 Flag-FWD1, 1 μg of flag-IKK1, and 5 μg of pCMV4 T7-IκBα. After 30 hours of transfection, cells were pretreated with 25 μmol/L MG132 for 1 hour, followed by 20 ng/ml tumor necrosis factor-α for 30 minutes. Cells were washed with ice-cold PBS once, lysed in RIPA buffer [20 mmol/L Tris-HCl, pH 7.4, 137 mmol/L NaCl, 10% (v/v) glycerol, 0.1% (w/v) SDS, 0.5% (w/v) deoxycholate, 1% (v/v) Triton X-100, 2.0 mmol/L EDTA, 1.0 mmol/L phenylmethylsulfonyl fluoride, 20 mmol/L leupeptin, 10 mmol/L N-ethyl maleimide (NEM), and 1% protease inhibitors], sonicated, cleared by centrifugation (13,000 rpm per minute for 30 minutes at 4°C), and immunoprecipitated with T7 tag antibody agarose (Novagen, Madison, WI). Immunoprecipitates were washed two times with RIPA buffer and two times with deubiquitination reaction buffers (50 mmol/L HEPES, pH 8.0, 01% Briji-35, and 3 mmol/L dithiothreitol). This yielded ubiquitinated IκBα, used as a substrate for deubiquitinase. To generate the ubiquitinated β-catenin (ub-β-catenin) substrates for the reaction, we treated human embryonic kidney 293T cells with the 25 μmol/L MG132 proteasome inhibitor for 2 hours, lysed cells with sonication in RIPA buffer, and immunoprecipitated β-catenin using anti-β-catenin antibodies. For the cell-free AvrA deubiquitination assay, we used both ubiquitinated IκBα (ub-IκBα) and ub-β-catenin as the substrates. Immunoprecipitated ub-T7-IκBα or ub-β-catenin was incubated with 1 μg of recombinant bacterial AvrA protein or point mutant C186A protein at 37°C in 50 μl of reaction buffer (50 mmol/L HEPES pH 8.0, 01% Briji-35, and 3 mmol/L dithiothreitol). Isopeptidase T (Boston Biochem, Cambridge, MA) served as positive control. Protease inhibitor N-ethyl maleimide (10 um) was added at the beginning of reaction. All reactions were terminated with 4× SDS loading buffer at different time points and boiled for 5 minutes before immunoblot analysis. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Invitrogen) following standard procedures.16Boone DL Turer EE Lee EG Ahmad RC Wheeler MT Tsui C Hurley P Chien M Chai S Hitotsumatsu O McNally E Pickart C Ma A The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses.Nat Immunol. 2004; 5: 1052-1060Crossref PubMed Scopus (893) Google Scholar We purified GST-AvrA and mutant AvrA C186A (mutated at the key cysteine required for its activity). We incubated ubiquitinated β-catenin with AvrA protein or AvrA C186A for 2 hours in reaction buffer at 37°C. Reactions were terminated by the addition of 6× SDS loading buffer. We analyzed the samples by SDS-polyacrylamide gel electrophoresis and Western blotting using an anti-ubiquitin antibody (Affiniti, Exeter, UK) and reprobed with an anti-β-catenin antibody. Tissues were fixed in 10% neutral buffered formaldehyde for 2 hours, transferred into 70% ethanol, and processed the next day by standard techniques.4Collier-Hyams LS Zeng H Sun J Tomlinson AD Bao ZQ Chen H Madara JL Orth K Neish AS Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-κB pathway.J Immunol. 2002; 169: 2846-2850PubMed Google Scholar Immunohistochemistry for apoptotic nuclei [terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining] was performed on paraffin-embedded sections (1 um) of mouse colons. Paraffin sections were baked in an oven at 56°C for 20 minutes. The slides were deparaffinized and rehydrated in xylene, followed by graded ethanol washes at room temperature. Antigen retrieval was achieved by boiling the slides in a microwave oven in 0.01 mol/L sodium citrate buffer, pH 6.0. Slides were then incubated in hydrogen peroxide (3% H2O2 and 1% fetal bovine serum in PBS) for 20 minutes at room temperature to block endogenous peroxidase activity, followed by incubation for 20 minutes in 5% fetal bovine serum/PBS to reduce nonspecific background. TUNEL staining was performed using the Apoptosis Detection Kit (ApopTag Plus Peroxidase In Situ; Chemicon International, Temecula, CA). A couple modifications were made to the kit's procedures: paraffin sections were baked (56°C for 20 minutes), and nuclei were counterstained with hematoxylin. The Automated Cellular Imaging System (ACIS; ChromaVision Medical Systems, San Juan Capistrano, CA) was used to quantitate TUNEL staining section according to the manufacturer's instructions. The number of proliferating cells was detected by immunoperoxidase staining for the thymidine analog bromodeoxyuridine (BrdU). 5-bromo-2′-deoxyuridine (100 mg/kg; Sigma) was injected i.p. 2 hours before sacrificing the mice. Specimens were fixed in 10% buffered formalin and handled as previously described in Materials and Methods. Slides were then incubated in 3% hydrogen peroxide for 20 minutes at room temperature to block endogenous peroxidase activity, followed by incubation for 20 minutes in a milk-peroxide solution (90 parts dH2O, five parts skim milk, and five parts 3% hydrogen peroxide) to reduce nonspecific background. The slides were incubated with polyclonal BrdU antibody (1:2000; RDI Divison of Fitzgerald Industries International, Concord, MA) for 1 hour at room temperature followed by biotinylated anti-sheep IgG (H+L) (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. Antibody staining was visualized with diaminobenzidine (Envision+ System/HRP Kit; DakoCytomation California Inc., Carpinteria, CA) and counterstaining with hematoxylin. Data are expressed as mean ± SD. Differences were analyzed by Student's t-test. P values <0.05 were considered significant. Previous work indicates that nonvirulent Salmonella PhoPc is able to attenuate NF-κB signaling by inhibiting IκBα degradation in vitro.3Neish AS Gewirtz AT Zeng H Young AN Hobert ME Karmali V Rao AS Madara JL Prokaryotic regulation of epithelial responses by inhibition of IκB-α ubiquitination.Science. 2000; 289: 1560-1563Crossref PubMed Scopus (747) Google Scholar To determine whether AvrA was responsible for the attenuation of NF-κB signaling by PhoPc, we focused on bacterial strains sufficient or deficient in AvrA: parental PhoPc, PhoPc AvrA mutant (AvrA−), or the AvrA complementary strain (PhoPc AvrA−/AvrA+). This system allowed us to concentrate on the cellular function of AvrA and exclude other bacterially induced effects on the host. In the human colonic epithelial cell line HCT116, parental PhoPc strain colonization inhibited IκBα degradation, whereas PhoPc AvrA− bacteria induced significant IκBα degradation. In cells colonized with PhoPc AvrA−/AvrA+, the complementary AvrA expression stabilized IκBα (Figure 1A, HCT116). The same response was also found in the rat small epithelial cell line IEC-18. Parental PhoPc strain colonization inhibited IκBα degradation, whereas PhoPc AvrA− bacteria induced more IκBα degradation. The complementary AvrA expression in PhoPcAvrA−/AvrA+ again stabilized IκBα (Figure 1A, IEC-18). With IκBα degradation, NF-κB is free to translocate to the nucleus. PhoPc AvrA− bacteria-infected cells had strong nuclear staining of NF-κB p65 (Figure 1B), whereas when AvrA was complemented back in PhoPc AvrA−/AvrA+, there was no NF-κB p65 translocation; this was comparable with the parental strain PhoPc, in which colonization did not induce translocation (Figure 1B), indicating AvrA expression regulated IκBα degradation and NF-κB distribution. These data are consistent with previous studies that AvrA expression attenuates NF-κB activity using the NF-κB transcriptional activity and IL-8 real-time PCR methods.4Collier-Hyams LS Zeng H Sun J Tomlinson AD Bao ZQ Chen H Madara JL Orth K Neish AS Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-κB pathway.J Immunol. 2002; 169: 2846-2850PubMed Google Scholar Taken together, our data suggest that AvrA expression in Salmonella strains is able to inhibit NF-κB activity in vitro. Using a streptomycin-pretreated mouse model, we further assessed IκBα expression and the NF-κB-dependent inflammatory cytokine IL-6 in vivo. As expected, infection with the parental PhoPc strain inhibited IκBα degradation, whereas PhoPc AvrA− bacteria induced more IκBα degradation in mouse colonic epithelial cells (Figure 2A). In mice infected with PhoPc AvrA−/AvrA+, AvrA expression stabilized IκBα (Figure 2A). Nonvirulent Salmonella parental PhoPc did not induce a large amount of IL-6 secretion, whereas the AvrA-deficient strain (AvrA−) increased IL-6 to 450 pg/ml. In contrast, AvrA overexpression (PhoPc AvrA−/AvrA+) was able to bring IL-6 to control levels (Figure 2B). Likewise, real-time PCR demonstrated that the absence of AvrA expression in PhoPc AvrA− increased IL-6 mRNA, whereas complementation with AvrA in PhoPc AvrA−/AvrA+ decreased IL-6 mRNA expression to the control levels similar to parental PhoPc(Figure 2C). Thus, the AvrA protein from S. typhimurium inhibits activation of the NF-κB pathway. The inhibitor of NF-κB, IκBα, is ubiquitinated before proteasomal degradation. Our data (Figure 1, Figure 2) and previous in vitro studies demonstrate that AvrA inhibits NF-κB activity by stabilizing IκBα.4Collier-Hyams LS Zeng H Sun J Tomlinson AD Bao ZQ Chen H Madara JL Orth K Neish AS Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-κB pathway.J Immunol. 2002; 169: 2846-2850PubMed Google Scholar One possibility is that AvrA might block the ubiquitinating E3 ligase such as β-TrCP.11Sun J Hobert ME Rao AS Neish AS Madara JL Bacterial activation of β-catenin signaling in human epithelia.Am J Physiol. 2004; 287: G220-G227Google Scholar However, we did not detect any protein level change of β-TrCP with AvrA expression in vitro (data not shown). Furthermore, we found that AvrA expression in the bacterial strain did not change the general ubiquitination of proteins detecting by Western blot (data not shown). Another possible mechanism of stabilizing IκBα would be removing ubiquitin from IκBα. It has been demonstrated that YopJ, a
Referência(s)