Human tumor necrosis factor alpha (TNFα) and human IL-1 were purchased from R&D Systems (Minneapolis, MN). Tris [hydroxymethyl]aminomethane (Tris) was purchased from Fisher Scientific (Fairlawn, NJ). Fetal calf serum was purchased from US Biotechnologies Inc. (Parkerford, PA). Para-nitro-phenylphosphate (PNPP) was purchased from Aldrich Chemical (Milwaukee, WI). The Polyacrylamide gel electrophoresis (PAGE) supplies: acrylamide, bis-acrylamide, sodium dodecyl sulfate (SDS), TEMED, and ammonium persulfate were purchased from Bio-Rad Laboratories (Hercules, CA). Dulbecco's modified essential medium (DMEM), DMEM:F12, phosphate buffered saline (PBS), glutamine, penicillin G, streptomycin, amphotericin B, and Grace's Insect medium were purchased from Invitrogen (Carlsbad, CA). Luria Broth (LB) was purchased from Becton Dickson and Co (Sparks, MD). The protease inhibitors: aprotinin, bestatin, leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Cal Biochem (La Jolla, CA). Protease inhibitor cocktail contained 10 μg/ml aprotinin, 2.5 μg/ml leupeptin, 8.3 μg/ml bestatin, and 1.7 μg/ml pepstatin A. Phorbol 12-myriState 13 acetate (PMA), N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes), anisomycin, and 2-[N-morpholino]ethanesulfonic acid (MES) were purchased from Sigma Chemical (St. Louis, MO). All other reagents were purchased from Sigma Chemical or Fisher Scientific unless Stated otherwise.
HT29 human intestinal (colorectal adenocarcinoma) epithelial cells (ATCC HTB-38), HeLa cervical epithelial adenocarcoma cells (ATCC CCL-2), 293T kidney cells (CRL-11268), A549 lung carcinoma cells (ATCC-185), and T98G glioblastoma cells (ATCC CRL-1690) were cultured in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO2atmosphere. T84 colorectal carcinoma cells (ATCC CCL-248) were cultured in DMEM:F12 with 2 mM glutamine, 5% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO2atmosphere. H5 insect cells (Invitrogen) were cultured in Grace's medium with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, 100 μg/ml Streptomycin, and 0.25 μg/ml amphotericin B at 28°C. MyD88-/-& TLR2-/-/TLR4-/-double knockout cells were obtained from Shizuo Akira and Osamu Takeuchi (Univ. of Osaka, Japan) and grown in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO2atmosphere.
Salmonella typhimuriumstrain SJW1103 (FliC, phase 1 flagellin, stabilized)  is a wild-typeSalmonella typhimuriumand can only express the Phase I fliC flagellin, SJW86 (SJW1103 FliC::TN10), and SJW134 (SJW1103 FliC and FljB deletions) were obtained from Robert Macnab (Yale Univ., Conn) and have been described .Salmonellaserovar dublin strain 2229, strain SE1 (2229 SopE mutant), strain SB2 (2229 SopB mutant), and SE1SB2 (2229 SopE and SopB mutant) were obtained from Edward Galyov (Compton Laboratory, Berkshire, UK) and have been described [14,15].Salmonellastrains for stimulation were grown in LB at 37°C without agitation for 16 hours, centrifuged at 6,000 × g for 1 minute, gently washed with PBS, and gently suspended in DMEM to maintain cells with attached flagella.
Plasmid pFM10.1 (ampicillin resistance), encodes a green fluorescent protein (GFP) expressed after theSalmonellahost is internalized by mammalian cells, obtained from Stanley Falkow (Stanford Univ., Stanford, CA) [10,34] and was transformed into strains SJW1103 and SJW134 by electroporation. Strains containing pFM10.1 were designated SJW1103G and SJW134G.
Preparation and analysis of Salmonella cell free culture supernatant
Native flagellin was harvested fromS. dublin2229 orS. typhimuriumSJW1103. Starter cultures were grown in Luria broth (LB) for 18 hours at 37°C with aeration, diluted 1:5000 in fresh LB, and grown for 12 hours under the same conditions. All subsequent procedures were performed at 4°C. Cells were removed from the medium by centrifugation at 10,000 × g for 5 min and discarded. The supernatant containing flagellin was filtered through a 0.8 micron filter (Millipore, Bedford, MA) to remove residual cells. Supernatant was concentrated 100 fold using an Amicon 100 kiloDalton (kDa) cutoff membrane (Millipore). Initial studies used concentrated culture supernatant fromS. dublinstrain 2229 that was treated with DNase, RNase, Protease K, boiled for 20 min or 100 mM DTT at 37°C for 2 hours and used for stimulation of cultured cells.
ConcentratedS. typhimurium1103 bacterial culture supernatant was washed 4 times by 1:10 dilution with 50 mM MES, pH 6.0, 50 mM NaCl and re-concentrated. Material not retained by the 100 kDa membrane was discarded. Washed culture supernatant was fractionated by gel permeation or anion exchange chromatography for analysis. For long-term storage, washed culture supernatant was supplemented with protease cocktail and stored at -20°C.
Fractionation by gel permeation chromatography was performed with a Superose 12HR column (Pharmacia) on a Bio-Logic system (Bio-Rad). One-half mililiter of 100× washed supernatant (equivalent of 50 ml original culture supernatant) was separated on the column at 0.4 ml/minute in 50 mM Hepes, pH 7.4, 200 mM NaCl. Fractions (0.5 ml) were collected, and 50 μl was fractionated by SDS-PAGE and stained with Bio-Safe Coomassie (Bio-Rad). Thirty microliters of each fraction was used for stimulation of HT29 cells (60 mm dishes) for 45 min and NF-κB DNA binding activity in the resulting whole cell extracts extracts were assayed by EMSA. The column was standardized with catalase (232 kDa), aldolase (158 kDa), abumin (67 kDa), ovalbumin (43 kDa), and Chymotripsinogen A (25 kDa), all obtained from Amersham-Pharmacia.
Fractionation by anion exchange chromatography was performed with Poros HQ matrix (2 ml column, PerSeptive Biosystems, Farmingham, MA) on a Bio-Logic system. Five mililiters of 100× washed supernatant (equivalent of 500 ml original culture supernatant) was separated at 1 ml/minute in 50 mM Hepes, pH 7.4, and a NaCl gradient from 50–500 mM. Fractions were collected and 5 μl of each fraction was examined by 10% SDS-PAGE. Proteins were fractionated on duplicate 10% SDS-PAGE precast gels (BioRad). One gel was stained with Bio-Safe Coomassie (Bio-Rad) and the protein bands were isolated for Mass Spectroscopy analysis (CCF Mass spectroscopy core facility) from the other identical non-stained gel, by electro-elution with a whole gel eluter (Bio-Rad) and SDS was removed with SDS-Out (Pierce, Rockland, IL) per the manufacturers directions. Proteins isolated from bands B1 to B6 were acetone precipitated by addition of 20 μg Aprotinin and 5 μg of BSA to each eluted fraction, ice-cold acetone (-20°C) was added to 80%, mixed well and precipitated overnight at -20°C. Proteins were pelleted by centrifugation at 14,000 × g in the cold for 30 min, acetone/liquid was removed and the pellets washed 2× with 1 ml acetone (-20°C). After removal of the acetone, protein pellets were air dried and then resuspended and denatured in 5 μl of 6 M guanidinium hydrochloride (Gu-HCl) at room temperature for 30 min. Resuspended proteins were two-fold serially diluted in DMEM to a final Gu-HCl concentration of 55 mM to renature the proteins. Two hundred fifty microliters of individual renatured proteins/DMEM were added per ml to HT29 cells (60 mm dishes) and whole cell extracts were prepared 45 min after stimulation and were assayed for NF-κB DNA binding activity by EMSA.
Purification of flagellin (purified flagellin)
The washed and concentrated culture supernatant fromS. typhimurium1103 containing flagellin was boiled for 20 minutes and precipitants removed by centrifugation at 15,000 × g. The supernatant containing flagellin was diluted 1:2 with 50 mM MES, pH 6.0, 50 mM NaCl and mixed with 2 ml Poros SP cation exchange matrix (PerSeptive Biosystems) per 1 liter of original culture. The Poros SP matrix was prepared as a 50% slurry and equilibrated with 50 mM MES, pH 6.0. The flagellin preparation and matrix were mixed on a roller at 12 to 14 RPM for 2 hours. The matrix along with bound contaminants was removed by filtration through a 0.85 micron filter and discarded, flagellin failed to bind to the cation exchange matrix at pH 6.0 and eluted in the flowthrough and was collected.
The pH of the flowthrough was adjusted by five-fold dilution of the sample with 50 mM Hepes, pH 7.8, 50 mM NaCl, and loaded onto a Poros HQ anion exchange column (2 ml column, PerSeptive Biosystems) equilibrated with 50 mM Hepes, pH 7.4, 50 mM NaCl. The column was washed with 2 volumes 50 mM Hepes, pH, 7.4, 50 mM NaCl, and eluted with a 10 column volume linear gradient of 50–500 mM NaCl in 50 mM Hepes, pH 7.4. Flagellin eluted from the column between 200–275 mM NaCl. Fractions containing flagellin were pooled and concentrated. The preparation was determined to be pure by electrophoresis of 5 μg protein by SDS-PAGE and stained with Bio-Safe Coomassie (Bio-Rad). Samples were stored at -80°C in 50 mM Hepes, pH 7.4, approx 225 mM NaCl, 10% glycerol and protease cocktail. A 4 liter preparation of culture supernatant yielded 2 mg purified flagellin.
In-gel tryptic digestion and protein identification by LC-MS
Gels were fixed and stained (Bio-Safe Blue, BioRad). All of the following procedures were performed by the CCF Mass spectroscopy core facility. Excised gel bands were reduced (100 mM DTT), and alkylated (100 mM iodoacetamide). Proteins in the gel bands were digested with modified trypsin (Promega, 20 μg/mL) with an overnight incubation at 37°C. Tryptic peptides were extracted from the gel with 50% acetonitrile, 0.1% acetic acid, concentrated in a SpeedVac (Thermo Savant) to remove acetonitrile, and reconstituted to 20 uL with 0.1% acetic acid. Extracted peptides were subjected to reversed phase (50 uM ID packed with Phenomenex Jupiter C18, 6 cm capillary column) liquid chromatography (2%–70% solvent B; Solvent A, 50 mM acetic acid, aqueous, Solvent B acetonitrile), coupled to a Finnigan LCQ DECA ion trap mass spectrometer for peptide sequencing, as described .
Preparation of GST-IκBa1-54 and GST-cJUN1-79 kinase substrates
IκBα amino acids 1 to 54 fused to GST or cJUN amino acids 1–79 fused to GST were prepared as previously described [37–39] and stored in kinase buffer (20 mM Hepes, pH 7.6, 10 MM MgCl2, 10 mM NaCl, 2 mM beta-glycerophosphate, 10 mM PNPP).
Preparation of cells for microscopy
HT29 cells for microscopic examination were grown in 6 well plates on sterile cover slips to a density of 50–75%. Cells were stimulated as described above. After stimulation, cover slips with HT29 cells were washed 2 times with ice cold PBS and fixed with 4% w/v formalin at room temperature for 20 minutes. Cells were washed 4 times with PBS prior to mounting for visualization ofSalmonellainvasion. Cover slips were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA), and cover slips sealed to slides.
Cells for antibody staining were treated with absolute methanol for 20 minutes following formalin fixation, then washed 3 times with PBS supplemented with 0.1% BSA (PBSB) and used directly or stored in the cold after azide was added to 0.02%. For p65(RelA) localization, cells on coverslips were blocked for 1 h at 37°C with PBS supplemented with 1% BSA. The PBSB was removed, washed once with PBSB and coverslips were placed cell-side down onto 150 μl of p65 antibody (Zymed, South San Francisco, CA) diluted 1:1500 in PBSB on a square of parafilm and placed in a humidified chamber at 37°C for 1.5 h. Coverslips were removed and placed cell-side up in 6-well dishes and washed 3 × 5 min with PBSB. Coverslips were then removed and placed cell-side down onto 150 μl of FITC-labeled donkey anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA) (1:300 in PBSB) on a square of parafilm and placed in a humidified chamber at 37°C for 1.5 h. Coverslips were removed and placed cell-side up in 6-well dishes and washed 5 × 5 min with PBSB, removed and placed cell-side down onto slides mounted with Vectashield (Vector Laboratories, Burlingame, CA) with DAPI and then sealed. NF-κB localization was determined by indirect immunofluorescence. Samples were observed on a Leica DMR upright microscope (Leica Microsystems Inc., Heidelberg, Germany) at 400× with oil immersion and equipped with FITC and UV filters. Images were collected with a MicroMax RS camera (Princeton Instruments Inc., Princeton, NJ), and Image Pro plus, version 4.5, software (Media Cybermetics Inc., Carlsbad, CA). Color enhancements were performed with Image Pro plus software. Visible light plus color overlays for Fig.1and Fig.5Bwere performed with MetaMorph Software (Universal Imaging Corp., Downington, PA).
Bacterial infection and cell stimulation
Mouse embryo fibroblasts (MEFs) or HT29 cells were grown in DMEM as above to a density of 90% prior to stimulation. All cells were washed with warm PBS and supplemented with DMEM without serum or antibiotics in preparation for stimulation. Cells were stimulated with; 10 ng/ml TNFα, 1 μg/ml flagellin unless specified otherwise, 20 μg/ml Anisomycin, 12.5 ng/ml PMA, or 108
Salmonella/ml at 37°C for desired times and extracts prepared as below. Cells harvested beyond one hour were washed with warm PBS and supplemented with warm DMEM, 2 mM glutamine, and 200 ug/ml gentamycin after 1 hour and returned to 37°C until extract preparation desired.
Whole cell extract preparation
Cells were washed with ice-cold PBS and all subsequent steps carried out at 4°C or on ice. Cells were scraped from the dish in ice-cold PBS, and collected by centrifugation at 1000 × g for 1 minute. Cells were lysed by suspension in 50 mM Tris-HCl, pH 7.6, 400 mM NaCl, 25 mM beta-glycerol phosphate, 25 mM NaF, 10 mM PNPP, 10 % glycerol, 0.5 mM sodium orthovanadate, 0.5% nonidet-40 (NP-40), 5 mM benzamidine, 2.5 mM metabisulfite, 1 mM PMSF, 1 mM DTT and protease inhibitor cocktail as described .
Electromobility shift assays (EMSA)
NF-κB DNA binding assays were carried out as previously described [3,35,38]. Anti-p65 antibody (Zymed, South San Francisco), anti-p50 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA), and anti-STAT3 antibody (Santa Cruz) were used for EMSA supershifts.
HT29 cells, 90–95% confluent in 35 mm round dishes, were prepared for stimulation as above and treated with a 1 ml suspension of Salmonella SJW1103 or SJW134 or left untreated in triplicate as above. After one hour, HT29 cells were washed 4× with warm PBS, supplemented with warm DMEM, 2 mM glutamine, and 200 μg/ml gentamycin, and incubated at 37°C for 4 hours. Cells were then harvested as above and lysed by suspension in 1 ml sterile distilled water. Ten-fold serial dilutions were prepared in PBS and 100 μl of each dilution was plated on LB agar plates and grown at 37°C for 20 hours. Colonies were counted and averaged.
Whole cell extracts (250 μg) were supplemented with 150 μl of Buffer A (20 mM Hepes, pH 7.9, 20 mM beta-glycerophosphate, 10 mM NaF, 0.1 mM orthovanadate, 5 mM PNPP, 10 mM 2-mercaptoethanol, 0.5 mM PMSF, and protease inhibitor cocktail), and immuno precipitation kinase assays carried out as described  using either IKKα monoclonal antibody (PharMingen – Becton Dickson), anti-JNK1 (Santa Cruz Biotechnologies, Santa Cruz, CA), or anti-hemagglutinin (HA) epitope antibody (Covence Antibodies, Princeton, NJ) as indicated. Protein G immunopellets were collected by centrifugation at 500 × g for 30 sec, washed 3 times with Buffer B (Buffer A plus 250 mM NaCl), and one time with Buffer C (Buffer A plus 50 mM NaCl and 10 mM MgCl2). Immunopellets were resuspended in 30 μl Kinase buffer with 0.1 mM orthovanadate, 50 μM "cold" ATP, 5 μCi γ-32P-ATP, 2 mM DTT, and 2 μg of soluble GST-IκBα1–54 or GST-cJUN1-79, and incubated at 30°C for 30 minutes. Reactions were stopped by the addition of 15 μl 4× SDS-PAGE loading buffer, heated at 95°C for 5 minutes, and resolved on 10% SDS-PAGE gels by standard procedures. Gels were rinsed, stained with Bio-Safe Coomassie (Bio-Rad) to visualize protein bands, rinsed, photographed then dried and exposed to Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY) to detect substrate phosphorylation.
Protein samples (40 μg) were resolved by SDS-PAGE on a 10% acrylamide gels by standard procedures, and proteins transferred to PVDF membrane (Millipore) and probed with antibodies as described . Membranes were washed 3× briefly with TBST, incubated with a 1:1000 dilution (1:800 for anti-TLR5) of the primary antibody in TBST, 1% non-fat milk for 1 hour, washed 3 × 5 min with TBST, and then incubated with a 1:2000 dilution of the appropriate HRP-conjugated secondary antibody in TBST, 0.5% non-fat milk for 1 hour. Primary antibodies used were: anti-IKKα/β (H-470, Santa Cruz), anti-JNK1, anti-ERK2 (K-23, Santa Cruz), anti-phospho-ERK (E-4, Santa Cruz), anti-p38MAPK (Cell Signaling Technologies, Beverly, MA), anti-phosopho-p38MAPK (Cell Signaling), anti-TLR5 (H-127, Santa Cruz), anti-muc1 (H-295, Santa Cruz) and anti-actin (C-11, Santa Cruz). Secondary antibodies used were: anti-mouse IgG HRP conjugate (Amersham-Pharmacia), anti-rabbit IgG HRP conjugate (Amersham-Pharmacia), anti-goat IgG-HRP conjugate (Santa Cruz). HRP activity was detected by ECL (Amersham-Pharmacia) as per manufacturers instructions, on Kodak X-OMAT AR film.
Construction of dominant-negative TLRs
All DN-TLRs were constructed using PCR. The universal 5' primer consisted of a 5'KPN I restriction site followed by sequences encoding the kozak sequence, translational start site, and preprotrypsin leader sequence of pCMV-1 (Sigma) that all the wild-type TLRs were initially cloned into. The 3' anti-sense (AS) primers were human TLRgene-specific primers (sequences available upon request) that created a stop codon immediately after a conserved tryptophan in Box 9 of the TLR TIR homology domain according to Bazan , thus creating carboxy terminus deletions. The 5' end of the AS primer contained a number of convenient restriction sites to allow directional cloning. PCR was performed with turbo-Pfu polymerase (Stratagene, La Jolla, CA) using standard procedures on individual wild-type TLR pCMV-1 plasmid DNAs (5 ng each, kind gifts of R. Medzhitov, Yale Univ. and R. Ulevitch, TSRI) [48,49] with the 150 ng each of the universal 5' sense primer and individual gene-specific TLR 3' primers. PCR products were cleaned-up with PCR cleanup kit (Qiagen, Germany) digested with appropriate restriction enzymes, gel purified and then ligated into the mammalian expression vector pCDNA3.1 (Invitrogen). Positive clones were sequenced to verify the mutations and tested for expression in transient expression assays and detected on immunoblots by probing with anti-FLAG M2 monoclonal antibody (Sigma). All wild-type and DN-TLR alleles are amino terminus FLAG epitope-tagged.
HT29 cells were transfected with Lipofectamine Plus (Invitrogen) as previously described . In transfections monitoring reporter gene expression, transfections were performed at least three times in 6 well dishes in triplicate with the total DNA mass kept constant at 4 μg (2 μg effector plasmid DNA, 100 ng 2× NF-κB Luc reporter gene, 50 ng pRL-TK, a thymidine kinase promoter drivenRenillaluciferase normalization reporter and 1.85 μg pCDNA3.1 plasmid DNA as bulk filler DNA) and fire-fly luciferase expression was normalized toRenillaluciferase expression using the dual-luciferase assay (Promega, Madison, WI). Fold inductions were calculated and values between experiments did not vary more than 15%, a representative experiment is presented. Transfection of 293T cells was performed with lipofectamine 2000 (Invitrogen) in 6-well dishes in triplicate as per the manufacture's protocol. TLR expression plasmids were added at 2 μg/well, and NF-κB and normalization control plasmids were as above with HT29 cells and pCDNA3.1 plasmid DNA as bulk filler DNA to a final DNA mass of 4 μg/well. Fold inductions were calculated and values between experiments (N of 3) did not vary more than 10%, a representative experiment is presented.
Real Reverse Transcription and Real Time PCR (RT2PCR)
Cells (N = 3) were stimulated 3 hours at 37°C with TNFα or FliC or left untreated and harvested for total RNA isolation. Total cellular RNA was extracted from cells with Trizol reagent (Invitrogen)  and reverse transcribed with ReactionReady first strand cDNA synthesis kit (SuperArray Bioscience Corp., Fredrick, MD). RNA (2.5 ug per 20 ul reaction) was reverse transcribed using random primers and Moloney murine leukemia virus reverse transcriptase per manufacturer specified conditions. Controls without reverse transcriptase (minus RT) was also generated for each RNA sample. RT2PCR was performed with an iCycler (Bio-Rad) to quantify TLR1 through TLR10 mRNA, 18S rRNA, and GAPDH mRNA. RT2PCR (25 ul reaction volume) was performed with the appropriate primers (SuperArray) per manufacturers instructions in triplicate with HotStart Taq DNA polymerase (SuperArray) at 95°C for 15 min to activate Taq and amplified for 40 cycles (95°C, 30 sec, 55°C, 30 sec, 72°C, 30 sec). RT2PCR was performed on the minus RT controls with TLR5 primers to detect DNA contamination. Real-time PCR analysis was performed using SYBR-green (Perkin-Elmer) according to manufacture's instructions with the specific primer pairs indicated above and primer pairs for 18S ribosomal RNA as reference RNA (Classic 18S primer pairs – Ambion Inc). Cycle time (Ct) was measured using the iCycler™ and its associated software (Bio-Rad). Relative transcript quantities were calculated by the ΔΔCt method using 18S ribosomal RNA as a reference amplified from samples using the Classic 18S primer pairs from Ambion, Inc (Austin, TX). Normalized samples were then expressed relative to the average ΔCt value for untreated controls to obtain relative fold-change in expression levels. Fold change in mRNA expression was expressed as 2ΔΔCt. ΔCt is the difference in threshold cycles for the TLR mRNAs and 18S rRNA. ΔΔCt is the difference between ΔCt non-simulated control and ΔCt stimulated sample. Values for fold-induction varied less than 5% among replicates.