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Infection and Immunity, March 2006, p. 1809-1818, Vol. 74, No. 3
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.3.1809-1818.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Conditioned Medium from Enterohemorrhagic Escherichia coli-Infected T84 Cells Inhibits Signal Transducer and Activator of Transcription 1 Activation by Gamma Interferon

Narveen Jandu,1,2,{dagger} Peter J. M. Ceponis,1,3,{dagger} Seiichi Kato,1,3 Jason D. Riff,1,3 Derek M. McKay,5 and Philip M. Sherman1,2,3,4*

Research Institute, Hospital for Sick Children,1 Institute of Medical Science,2 Departments of Laboratory Medicine and Pathobiology,3 Paediatrics, University of Toronto, Toronto, Canada,4 Intestinal Disease Research Programme, McMaster University, Hamilton, Canada5

Received 14 September 2005/ Returned for modification 24 October 2005/ Accepted 12 December 2005


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ABSTRACT
 
Gamma interferon (IFN-{gamma}) is a cytokine important to host defense which can signal through signal transducer and activator of transcription 1 (Stat1). Enterohemorrhagic Escherichia coli (EHEC) modulates host cell signal transduction to establish infection, and EHEC serotypes O113:H21 and O157:H7 both inhibit IFN-{gamma}-induced Stat1 tyrosine phosphorylation in vitro. The aim of this study was to delineate both bacterial and host cell factors involved in the inhibition of Stat1 tyrosine phosphorylation. Human T84 colonic epithelial cells were challenged with direct infection, viable EHEC separated from T84 cells by a filter, sodium orthovanadate, isolated flagellin, bacterial culture supernatants, and conditioned medium treated with proteinase K, trypsin, or heat inactivation. Epithelial cells were then stimulated with IFN-{gamma} and protein extracts were analyzed by immunoblotting. The data showed that IFN-{gamma}-inducible Stat1 tyrosine phosphorylation was inhibited when EHEC adhered to T84 cells, but not by bacterial culture supernatants or bacteria separated from the epithelial monolayer. Conditioned medium from T84 cells infected with EHEC O157:H7 suppressed Stat1 activation, and this was not reversed by treatment with proteinases or heat inactivation. Use of pharmacological inhibitors showed that time-dependent bacterial, but not epithelial, protein synthesis was involved. Stat1 inhibition was also independent of bacterial flagellin, host proteasome activity, and protein tyrosine phosphatases. Infection led to altered IFN-{gamma} receptor domain 1 subcellular distribution and decreased expression in cholesterol-enriched membrane microdomains. Thus, suppression of host cell IFN-{gamma} signaling by production of a contact-dependent, soluble EHEC factor may represent a novel mechanism for this pathogen to evade the host immune system.


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INTRODUCTION
 
Enterohemorrhagic Escherichia coli (EHEC) refers to a family of bacterial enteropathogens that can contaminate food and water to cause outbreaks of diarrhea and hemorrhagic colitis (10, 25). In addition, the Shiga-like toxins expressed by EHEC have been associated with causing hemolytic uremic syndrome, a leading cause of acute renal failure among the pediatric population (10, 56). Multiple serotypes of EHEC, including O157:H7 and non-O157 serotypes, elicit these diseases. Indeed, non-O157 serotypes are increasingly recognized as important human enteropathogens (23), although they often lack the well-characterized virulence factors present in O157:H7 strains (12). Non-O157:H7 EHEC, such as serotype O113:H21, are generally negative for the locus for enterocyte effacement (LEE) pathogenicity island and therefore do not express the outer membrane protein intimin or a functional type III secretion system. However, both O157:H7 and O113:H21 express Shiga-like toxins (26, 13), indicating these pathogens can utilize both similar and divergent strategies to infect host cells. These data highlight a need to better understand the different EHEC serotypes and how they interact with the enterocyte.

Cholesterol-enriched microdomains in the lipid bilayer are biochemically distinct regions of the eukaryotic plasma membrane and also contain sphingolipids and proteins important in signal transduction (48). Such microdomains provide a platform for host cell signal transduction cascades that initiate from an extracellular stimulus (33). For instance, functional gamma interferon (IFN-{gamma}) receptor (IFNGR) domains 1 and 2 reside in membrane microdomains of epithelial cells, and pharmacological disruption of microdomains inhibits IFN-{gamma}-induced signal transducer and activator of transcription 1 (Stat1) tyrosine phosphorylation in the cytosol and DNA binding in the nucleus (40, 51, 52). In addition to serving as platforms for host-driven signal transduction, bacteria and their products manipulate membrane microdomains as part of their pathogenic strategy (31, 38). For example, when intimately attaching to host cells, enteropathogenic E. coli causes the clustering of microdomain-associated host cell proteins (59). Epithelial cell vacuolization by the vacuolating cytotoxin A of H. pylori occurs in a microdomain-dependent manner (39). However, whether bacterial infection can also target microdomains to disrupt components of host cell signal transduction cascades, such as cytokine receptors, is largely unknown.

IFN-{gamma} plays a central role in the Th1 cellular immune response against a wide variety of microbes (44). Citrobacter rodentium is a murine attaching and effacing pathogen model for EHEC O157:H7 infection that elicits a Th1-response typified by IFN-{gamma} production (19). Indeed, IFN-{gamma} knockout mice infected with C. rodentium demonstrate worse symptoms of disease than their wild-type littermates (47). Previously, we showed that infection with EHEC O157:H7 and O113:H21 disrupts IFN-{gamma}-induced Stat1 signal transduction in epithelial cells in vitro (4). These inhibitory effects were independent of the locus of enterocyte effacement pathogenicity island (PAI), the type III secretion system, Shiga-toxins type 1 and type 2, and the pO157 plasmid (4). Moreover, live bacteria, but not heat-killed organisms, are required to elicit the inhibition of IFN-{gamma}-stimulated Stat1-tyrosine phosphorylation. Taken together, these observations suggest that IFN-{gamma}-Stat1 signal transduction likely is important to host defense in response to attaching-effacing bacterial enteropathogens.

The objectives of this study were to further delineate both bacterial and host cell factors that mediate EHEC suppression for IFN-{gamma}-induced Stat1 signal transduction. The data presented herein suggest a role for EHEC adhesion, bacterial protein synthesis, and a contact-dependent soluble mediator in suppression of IFN-{gamma} signal transduction. In addition, EHEC infection disrupts the subcellular localization of IFN-{gamma} receptor 1 in human epithelial cells.


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MATERIALS AND METHODS
 
Eukaryotic tissue culture. T84 colonic epithelial cells were cultured as previously described (4). Briefly, cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 10% (vol/vol) fetal bovine serum (FBS), 2% penicillin-streptomycin, 2% sodium bicarbonate, and 0.6% L-glutamine (all obtained from Life Technologies, Grand Island, N. Y.). Approximately 3 x 106 T84 cells were seeded onto 6-cm petri dishes (Falcon) or six-well plates (Costar) and grown to confluence for whole-cell protein extraction. Cells were grown at 37°C in a 5% CO2 atmosphere. Prior to bacterial infection or protein extraction, the cells were incubated in medium containing no antibiotics for ~20 h.

Bacteria and bacterial growth conditions. EHEC of serotypes O157:H7 (strain CL-56) and O113:H21 (strain CL-15) were kept on 5% sheep blood agar plates at 4°C and cultured in static, nonaerated Penassay broth (Difco Laboratories, Detroit, MI) overnight at 37°C (4, 22). EHEC serotype O157:NM (NM, nonmotile), including strain E32511, confirmed not to express flagellin (13), and strain MOH67 were grown under the same conditions (kind gifts from M. A. Karmali, Health Canada, Guelph, Ontario, Canada). To infect epithelial cells, bacteria were pelleted from broth cultures by centrifugation at 3,000 rpm for 5 min and washed in cell culture medium. The bacteria were resuspended in a final volume of 25 µl of tissue culture medium without antibiotics and then added to host cells, where the multiplicity of infection (MOI) was 100 bacteria per epithelial cell (4). The same volume of medium alone served as a vehicle control treatment. In some experiments, bacteria in tissue culture medium were physically separated from T84 epithelial cells grown on the bottom of a six-well tissue culture plate by placing the bacteria in a compartment above the cells, separated by a 0.4-µm Transwell filter (Costar). Medium from the T84 compartment beneath the filter was cultured on 5% sheep blood agar plates to ensure lack of bacterial growth.

Bacterial culture supernatants and conditioned medium. To collect bacterial culture supernatants, 1 ml of EHEC O157:H7 grown overnight in Penassay broth (10 ml, static) was centrifuged (3,000 rpm, 5 min) and resuspended in 15 ml of T84 tissue culture medium with FBS and without antibiotics. After 6 h growth at 37°C in a 5% CO2 atmosphere, the medium was centrifuged (3,000 rpm, 15 min), filtered (0.45 µm) and stored at –20°C. To collect conditioned medium, EHEC O157:H7 strain CL-56 grown overnight in 10 ml Penassay broth (37°C, static) was used to infect confluent monolayers of T84 cells (MOI 100:1, 6 h), while T84 cells receiving medium only served as a control. After 6 h, medium was cleared by centrifugation (3,000 rpm, 15 min), filtered (0.45 µm), and stored at –20°C until use. This conditioned medium contained factors secreted either by T84 cells alone or by bacterium-T84 cell contact. In both cases, the effectiveness of filtration was confirmed by lack of bacterial growth from 100 µl of this conditioned medium plated onto 5% sheep blood agar plates and then incubated overnight at 37°C.

Whole-cell protein extraction. T84 cells were either infected with Escherichia coli or incubated with conditioned medium. Subsequently, some cells were stimulated with IFN-{gamma} (50 ng/ml) at 37°C for 30 min. Next, T84 cells were washed three times with ice-cold phosphate-buffered saline (PBS) (pH 7.4) and the whole-cell protein extracts were collected and stored at –80°C as previously described (4).

Isolation of EHEC O157:H7 flagellin. EHEC flagellin was isolated as previously described (2, 43). Briefly, overnight cultures of EHEC O157:H7 strain CL-56 were resuspended in a hydrochloric acid (pH 2.0) solution containing 1.0 mM phenylmethylsulfonyl fluoride for 30 min at room temperature. Bacteria were then pelleted at 5,000 x g for 30 min. Protein from the flagellin-rich supernatant was concentrated using a Millipore centrifugal filter device (5-kDa cutoff), protein concentration was determined by the Bio-Rad assay, and samples were stored at –80°C until use. An aliquot (5 or 10 µg) of the sample was electrophoresed through a 10% Tris-HCl gel (Bio-Rad), and the gel subsequently Coomassie brilliant blue-stained to confirm that the flagellin subunit (approximately 66 kDa) was successfully isolated (43).

Pharmacological inhibitor studies. T84 cells were preincubated with the eukaryotic cell protein synthesis inhibitor cycloheximide (10 µg/ml, 45 min) (57), with inhibitor remaining in the medium throughout the infection period. In addition, epithelial cells and bacteria were incubated with the bacterial protein synthesis inhibitor chloramphenicol (100 µg/ml) at 0, 30, 90, and 180 min after infection with EHEC O157:H7 (MOI 100:1, 6 h) (4). MG-132 (50 µM; preincubation for 45 min, left in the medium for the infection period) was employed to determine the role of the host proteasome (42). Sodium orthovanadate (1,000 µM for the entire 6 h infection or for 30 min following infection) treatment of infected and uninfected T84 cells was used to determine the effects of protein tyrosine phosphatases (41). In all cases, the effects of vehicle alone were also evaluated and found to have no effect on the tyrosine phosphorylation status of Stat1 or expression levels of native Stat1 (data not shown).

Infected and uninfected T84 cells were also coincubated with either the quorum sensing mimetic norepinephrine (10 to 500 µM) (49) or the quorum sensing inhibitor propranolol (10 to 250 µM) (49) at 37°C for 3.5 h to determine the role of quorum sensing in the ability of EHEC O157:H7 to suppress IFN-{gamma}-induced Stat1 tyrosine phosphorylation.

Proteinase and heat inactivation treatment of conditioned medium. Conditioned medium from T84 cells infected with EHEC O157:H7 strain CL-56 was incubated with proteinase K (200 µg/ml, 1 h, 37°C). Subsequently, proteinase K activity was neutralized by adding the serine protease inhibitor Pefabloc (Roche/Boehringer) (5 mM, 2 h, 37°C). In addition, trypsin digestion of conditioned medium was similarly performed, except that FBS-free T84 culture medium was studied. Conditioned medium from T84 cells infected with EHEC O157:H7 strain CL-56 was employed. Following incubation with trypsin (200 µg/ml, 1 h, 37°C) in the medium, soybean trypsin inhibitor (400 µg/ml, 30 min, 37°C) was added to neutralize proteinase activity (15). Such medium was also prepared with the addition of 10% FBS.

Conditioned medium from T84 cells alone and from T84 cells infected with EHEC O157:H7 strain CL-56 was heat inactivated by boiling (100°C, 2 h). Subsequently, conditioned medium was cooled to 37°C and employed in protein extraction experiments.

Immunoblotting. Equal volumes of whole-cell protein extracts were analyzed for tyrosine phosphorylated Stat1 and native Stat1, as previously described (4) or with the following modifications: sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading buffer was added to whole-cell protein extracts in a 1:2 (vol/vol) ratio, samples were boiled for 3 min and then loaded into wells of precast 10% polyacrylamide gels (Ready Gel; Bio-Rad Laboratories, Hercules, CA). Gels were electrophoresed at 111 V for 1 to 1.5 h, and migrated proteins were then transferred onto nitrocellulose membranes (BioTrace NT; Pall Corporation, Ann Arbor, MI) at 100 V for 1.5 to 2 h at 4°C. The nitrocellulose membranes were subsequently incubated in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h at 20°C. The blocking buffer was decanted and the membranes were incubated with either anti-native Stat1 primary antibody (Santa Cruz Biotechnologies, Santa Cruz, CA; 1:5,000 dilution in Odyssey buffer) or anti-phospho-Stat1 primary antibody (Cell Signaling, Beverly, MA; 1:1,000 dilution) and anti-ß-actin primary antibody (1:5,000 dilution; Sigma) at 4°C overnight on a shaker. Primary antibody solutions were subsequently decanted and the membranes were washed four times with PBS plus 0.1% Tween (5 min per wash).

The membranes were then incubated with secondary antibodies: IRDye 800 goat anti-rabbit immunoglobulin G (IgG) (1:20,000 dilution; Rockland Immunochemicals, Gilbertsville, PA) and Alexa Fluor 680 goat anti-mouse IgG (1:20,000 dilution; Molecular Probes, Eugene, OR) at 20°C for 1 h on a shaker. Nitrocellulose membranes were again washed four times with PBS-Tween with a final wash in PBS without Tween. Immunoblots were also probed with anti-Jak1 (AnaSpec, San Jose, CA; 1:1,000 dilution) and anti-Jak2-phosphospecific (pTyr10097/1008) (AnaSpec; 1:1,000 dilution) antibodies to determine if the inhibitory activity occurred upstream of Stat1-phosphorylation (45). Bands were detected by scanning the nitrocellulose membranes into the Odyssey system (LI-COR Biosciences) with both 700-nm and 800-nm channels, at a resolution of 169 µm (58).

Densitometry. The integrative intensity of all bands was calculated using automated software provided with the Odyssey Infrared Imaging System (LI-COR Biosciences). For immunoblots with dual antibody staining, integrative intensity values were normalized to the integrative intensities obtained for either anti-native Stat1 or ß-actin (58). Normalized values obtained from bands of uninfected cells stimulated with IFN-{gamma} were then set to 100%, with samples derived from EHEC-infected cells then calculated as a percentage relative to uninfected stimulated cells.

Localization of IFN-{gamma} receptor 1 to microdomains using buoyant density ultracentrifugation. T84 cells were grown to confluence in 3-cm tissue culture dishes and detergent resistant microdomains were isolated, as described previously (51). After a 6-h infection period, T84 cells were washed three times in ice-cold PBS, scraped with a rubber policemen, pipetted into 15-ml conical tubes, and centrifuged at 1,000 rpm for 2 min. Pelleted cells (~106) were resuspended in 0.8 ml of extraction buffer (25 mM Tris-HCl, 150 mM NaCl, 10% sucrose, 1% Triton X-100, 1 mM dithiothreitol, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, 10 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate; all from Sigma Aldrich, Oakville, Ontario, Canada). The lysate was then passed through a 25-gauge needle 10 times and incubated for 30 min on ice.

Optiprep stock solution (Invitrogen, Burlington, Ontario, Canada) (60%) was then added to cell lysates (2:1, vol/vol); 2.5 ml of the 40% solution, loaded into the bottom of a 12-ml ultracentrifuge tube (catalogue 344059; Beckmann, Palo Alto, CA), and then overlaid with 6.5 ml of 35% and 3.0 ml of 5% Optiprep solutions, diluted in extraction buffer. Samples were centrifuged for 20 h (at 36,000 rpm) at 160,000 x g at 4°C in an SW41 Ti rotor (Beckman). After ultracentrifugation, eight 1.5-ml fractions were collected and snap-frozen in liquid nitrogen for storage at –80°C, prior to analysis by immunoblotting (4, 51). For Western blotting, membranes were incubated with polyclonal rabbit anti-human antibody against IFNGR1 (1:200; C-20) or caveolin-1 (1:500; N-20) (all from Santa Cruz). Immunoblots were then washed and incubated with secondary antibody, IRDye 800 goat anti-rabbit IgG (1:10,000 dilution; Rockland Immunochemicals).

Confocal microscopy. T84 cells grown on glass coverslips were washed three times with ice-cold PBS (pH 7.4), fixed in 4% paraformaldehyde for 30 min, permeabilized for 4 min with 0.1% Triton X-100, and blocked for 30 min in 2% bovine serum albumin/0.1% Triton X-100 (4, 28). Subsequently, cells were probed for IFNGR1 with 1:100 anti-IFNGR1 (Santa Cruz) in 2% bovine serum albumin overnight at 4°C, washed in PBS, incubated with 1:500 fluorescein isothiocyanate-red goat anti-Armenian hamster antibody (Jackson Labs) in bovine serum albumin/Triton X-100 for 1 h at room temperature, and washed in PBS (pH 7.4). Vectashield (Vector Labs, Burlington, Ontario, Canada) mounting medium for fluorescence was added, and slides were sealed with coverslips and examined using a confocal microscope (Leica DMIRE2 spinning disk confocal microscope).


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RESULTS
 
Live EHEC must be in contact with epithelial cells to suppress Stat1 activation. Adhesion of microbes to host cells can be integral to causing disease (14). Our previous studies showed that live, but not heat-killed, EHEC O157:H7 suppressed Stat1 activation (4). Here we determined whether direct contact between live EHEC serotype O157:H7 or O113:H21 and epithelial cells was required. Analysis of whole-cell protein extracts showed that IFN-{gamma} induced tyrosine phosphorylation of Stat1 in uninfected T84 cells, whereas infection with EHEC O157:H7 or O113:H21 in the lower chamber resulted in complete inhibition of Stat1 tyrosine phosphorylation (Fig. 1). In contrast, when either strain was placed in the upper chamber, separated from the T84 monolayer by insertion of a Transwell filter, Stat1 activation was not inhibited. These data suggest that direct contact between the bacterium and epithelial cell is required to inhibit Stat1 activation.


Figure 1
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  		FIG. 1. EHEC must contact host cells to suppress IFN-{gamma}-induced Stat1 tyrosine phosphorylation. T84 cells were grown in a six-well plate, and a Transwell filter (0.4 µm) was placed above the cells before infection. Subsequently, cells were infected with EHEC (MOI 100:1, 6 h), rinsed with PBS, and stimulated with IFN-{gamma} (50 ng/ml, 30 min), and whole-cell protein extracts were analyzed by immunoblotting. (A) IFN-{gamma} induces Stat1 tyrosine phosphorylation compared with uninfected, untreated T84 cells (upper panel). Infection with EHEC O157:H7 strain CL-56 placed in the basolateral chamber directly on the T84 cells suppressed the ability of IFN-{gamma} to stimulate Stat1 tyrosine phosphorylation (basal chamber CL-56). In contrast, EHEC O157:H7 placed in the upper chamber (apical chamber CL-56) that was separated from the T84 cells by a Transwell filter did not prevent subsequent stimulation of Stat1 tyrosine phosphorylation by IFN-{gamma} (n = 3). Total levels of native Stat1 were approximately equivalent between lanes (Lower panel). (B) Similarly, EHEC O113:H21 strain CL-15 only suppressed IFN-{gamma}-induced Stat1 tyrosine phosphorylation when placed in the basolateral chamber (n = 3). Lower panel, native Stat1.

Conditioned medium, but not bacterial culture supernatant, suppresses IFN-{gamma}-induced Stat1 tyrosine phosphorylation. To confirm the necessity of direct contact between bacteria and epithelial cells in suppressing IFN-{gamma}-induced Stat1 activation, T84 cells were incubated with EHEC O157:H7 bacterial culture supernatant, or with conditioned medium taken either from T84 cells alone or from T84 cells infected with EHEC O157:H7. Both T84 cells treated with bacterial culture supernatant alone or with T84 media alone still supported IFN-{gamma} activation of Stat1 tyrosine phosphorylation (Fig. 2A). In contrast, incubation of T84 cells with conditioned medium from tissue culture cells infected with EHEC O157:H7 resulted in reduced (16% of control) activation of Stat1 tyrosine phosphorylation by IFN-{gamma} relative to uninfected epithelial cells (Fig. 2B). These results indicate that a soluble factor(s) generated from contact between bacteria and epithelial cells, but not from bacteria or epithelial cells alone, suppresses Stat1 activation. Also, these data complement the results shown in Fig. 1 that direct contact between EHEC and the epithelial cell is necessary to interrupt Stat1 activation.


Figure 2
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  		FIG. 2. Conditioned medium from T84 cells infected with EHEC O157:H7 suppresses IFN-{gamma}-induced Stat1 tyrosine phosphorylation. T84 cells grown in 6-cm dishes were treated with either bacterial culture supernatant (CS; 100%, 6 h) or conditioned medium (CM; 100%, 6 h), rinsed with PBS, and stimulated with IFN-{gamma} (50 ng/ml, 30 min) and whole-cell protein extracts were analyzed by immunoblotting. (A) T84 cells exposed to T84 medium alone (lanes 1 and 2) or to culture supernatant of EHEC O157:H7 strain CL-56 (100% CS) both supported activation of Stat1 tyrosine phosphorylation after stimulation with IFN-{gamma} (upper panel). Total native Stat1 protein levels were approximately equal between lanes (lower panel) (n = 3). (B) Conditioned medium from T84 cells alone supported Stat1 activation (lane 2), whereas treatment with conditioned medium taken from T84 cells infected with EHEC O157:H7 strain CL-56 (100% CM) reduced IFN-{gamma}-induced Stat1 tyrosine phosphorylation (upper panel). Lower panel, total levels of native Stat1 protein (n = 4).

Soluble factor(s) is insensitive to proteinase and heat inactivation treatment. To determine if the soluble factor(s) responsible for suppression of IFN-{gamma}-induced Stat1 activation was a peptide or nonpeptide, we incubated conditioned medium, taken from T84 cells only or T84 cells infected with EHEC, with either proteinase K or trypsin. Subsequently, proteinase K activity was neutralized by adding the serine protease inhibitor Pefabloc to prevent cytotoxicity from the protease, while trypsin was neutralized using soybean trypsin inhibitor. Results indicate that proteinase K treatment did not reverse the suppressive effect of conditioned medium from T84 cells infected with EHEC O157:H7 on IFN-{gamma}-induced Stat1 tyrosine phosphorylation (Fig. 3A). Similarly, treatment with trypsin and the soybean trypsin inhibitor did not reverse the inhibition of IFN-{gamma}-induced Stat1 tyrosine phosphorylation by conditioned medium (Fig. 3B). These results suggest that the factor responsible for suppression of Stat1 activation is proteinase insensitive.


Figure 3
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  		FIG. 3. Effect of conditioned medium treated with proteinases or with heat inactivation. Whole-cell protein extracts from T84 cells grown in 6-cm dishes were analyzed by immunoblotting. (A) Conditioned medium from T84 cells alone supported Stat1 tyrosine phosphorylation stimulated by IFN-{gamma} (50 ng/ml, 30 min) (upper panel) (lane 3). In contrast, conditioned medium from T84 cells infected with EHEC O157:H7 strain CL-56 partially suppressed IFN-{gamma}-induced Stat1 tyrosine phosphorylation (lane 4). The suppressive effect of this conditioned medium was not reversed by preincubation with proteinase K (PK) (200 µg/ml, 1 h, 37°C) and subsequent neutralization of PK, to prevent cytotoxicity, with Pefabloc (PF) (5 mM, 2 h, 37°C) (n = 2). Lower panel, total Stat1 levels. (B) Similarly, the suppressive effect of conditioned medium from T84 cells infected with EHEC O157:H7 on Stat1 activation was not reversed by preincubation with trypsin (200 µg/ml, 1 h, 37°C) and subsequent neutralization with soybean trypsin inhibitor (SB) (400 µg/ml, 30 min, 37°C) (n = 2). Lower panel, total Stat1 levels. (C) Conditioned medium from T84 cells alone or from T84 cells infected with EHEC O157:H7 was boiled for 2 h and then incubated with T84 cells (6 h, 37°C) before IFN-{gamma} treatment (50 ng/ml, 30 min). IFN-{gamma}-induced Stat1 activation was partially suppressed by conditioned medium from EHEC O157:H7-infected T84 cells, and heat inactivation treatment did not reverse this suppressive effect (upper panel, lanes 4 and 5) (n = 2). Lower panel, total Stat1 activity.

In addition, conditioned medium taken from T84 cells with EHEC O157:H7 strain CL-56 was heat inactivated and then incubated with T84 cells. Heat-inactivated conditioned medium from T84 cells infected with EHEC still suppressed IFN-{gamma}-induced Stat1 activation (Fig. 3C). Together with the proteinase data, this finding suggests that the factor(s) responsible for suppressing IFN-{gamma}-induced Stat1 tyrosine phosphorylation is nonpeptide.

Bacterial but not epithelial cell protein synthesis mediates suppression of Stat1 activation. Bacterial infection can induce expression of suppressors of cytokine signaling (SOCS) in host cells, which can then suppress subsequent cytokine signal transduction (8, 50). Thus, we tested the role of bacterial and host cell protein synthesis during Stat1 inhibition. First, inhibition of bacterial protein synthesis by coincubation with chloramphenicol completely blocked the ability of EHEC O157:H7 to suppress IFN-{gamma}-induced Stat1 tyrosine phosphorylation (Fig. 4A). This effect was time dependent, as addition of chloramphenicol more than 30 min after infection was unable to reverse the inhibition of Stat1 tyrosine phosphorylation by EHEC. In contrast, inhibition of host cell protein synthesis using cycloheximide did not block the ability of EHEC O157:H7 infection to suppress Stat1 activation (Fig. 4B). Therefore, time-dependent bacterial but not host cell protein synthesis is required to suppress Stat1 activation by IFN-{gamma}.


Figure 4
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  		FIG. 4. Bacterial, but not epithelial cell, protein synthesis mediates suppression of Stat1 activation. T84 cells were grown in 6-cm dishes, treated with an inhibitor, and infected with EHEC O157:H7 strain CL-56 (6 h, 37°C). Subsequently, epithelial cells were rinsed with PBS and stimulated with IFN-{gamma} (50 ng/ml, 30 min) and whole-cell protein extracts were analyzed by immunoblotting. (A) T84 cells alone or those infected with EHEC O157:H7 were incubated with the bacterial protein synthesis inhibitor chloramphenicol (Cph) (100 µg/ml; coincubation to addition 180 min after infection). Chloramphenicol time-dependently blocked the EHEC-mediated suppression of IFN-{gamma}-induced Stat1 activation, and had no effect alone on Stat1 activation. Total Stat1 levels are shown in the lower panel (n = 3). (B) T84 cells were preincubated with the host cell protein synthesis inhibitor cycloheximide (10 µg/ml, 45 min), and inhibitor remained in the medium during infection. Preincubation with cycloheximide did not block the ability of EHEC O157:H7 to suppress IFN-{gamma}-induced Stat1 tyrosine phosphorylation (upper panel) (n = 3). The lower panel shows total Stat1 levels.

Mechanisms of EHEC-mediated suppression of Stat1 activation. Stat activity is down-regulated by the host cell proteasome (37). Also, proteasome inhibition can prevent NF-{kappa}B activation, and NF-{kappa}B activity is implicated in suppression of Stat1 activation (27) and SOCS expression (32). Preincubation with the proteasome inhibitor MG-132 did not reverse the ability of EHEC to suppress IFN-{gamma}-induced Stat1 tyrosine phosphorylation, indicating that the effect is proteasome-independent (Table 1).


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  		TABLE 1. Mechanisms of EHEC-mediated suppression of Stat1 activation

Protein tyrosine phosphatases are known to deactivate phosphorylated Stat1 (18, 53). To determine if the inhibition of Stat1-tyrosine phosphorylation by EHEC O157:H7 was due to either a bacterial protein tyrosine phosphatase or the upregulation of a host cell protein tyrosine phosphatase, infected and uninfected T84 cells were treated with sodium orthovanadate. Since a phosphorylated Stat1 band was not recovered in cell extracts of EHEC-infected cells, the inhibitory effects of EHEC O157:H7 on Stat1-tyrosine phosphorylation likely is not due to a bacterial protein tyrosine phosphatase or the up-regulation of a host cell protein tyrosine phosphatase (Table 1).

Bacterium-mediated mechanisms. Activation of Toll-like receptors can suppress cytokine signal transduction in macrophages (8), and T84 cells express the functional receptor for flagellin, TLR5 (2). However, treatment of T84 cells with flagellin isolated from EHEC O157:H7 did not inhibit subsequent IFN-{gamma}-induced Stat1 activation (Table 1). Furthermore, infection of T84 cells with two nonmotile EHEC O157:H- strains, E32511 and MOH67, also resulted in suppression of IFN-{gamma}-induced Stat1 tyrosine phosphorylation (Table 1). These data indicate that EHEC suppression of cytokine signaling does not require expression of flagella and reinforce the NF-{kappa}B-independent nature of Stat1 inhibition.

Quorum sensing by EHEC O157:H7 regulates expression of multiple EHEC virulence factors, including type III secretion and attaching-effacing lesion formation on host cells (49). To determine if quorum sensing was involved in the EHEC-mediated suppression of Stat1 activation, we coincubated T84 cells with either propanolol, an inhibitor of quorum sensing, or norepinephrine, a quorum-sensing mimetic (49). Norepinephrine did not enhance the ability of EHEC O157:H7 to suppress IFN-{gamma}-induced Stat1 tyrosine phosphorylation. Moreover, propanolol did not reverse the EHEC-mediated inhibition of Stat1 activation. Taken together, these data suggest that expression of the bacterial factor(s) responsible for suppressing Stat1 activation is not regulated by EHEC quorum sensing (Table 1).

EHEC O157:H7 infection disrupts Jak phosphorylation. IFN-{gamma} signals through the IFN-{gamma} receptor, a cell surface receptor comprised of two functional domains: IFN-{gamma} receptor domain 1 provides the binding site for IFN-{gamma}, while IFN-{gamma} receptor domain 2 initiates downstream signaling events (1). The cytoplasmic tails of both domains are linked to Jak proteins, which upon phosphorylation provide docking sites for latent Stat1 proteins. Jak1 is linked to IFN-{gamma} receptor domain 1 and Jak2 is linked to IFN-{gamma} receptor domain 2 (1). To determine if EHEC O157:H7 inhibition of Stat1 phosphorylation occurs upstream of Stat1, cell extracts of infected and uninfected T84 cells were probed with an anti-Jak1 and an anti-Jak2 phosphospecific (pTyr10097/1008) antibody (Fig. 5). Relative to uninfected cells, the constitutive amounts of Jak1 and phospho-Jak2 in EHEC-infected cells were 67 and 96%, respectively, as determined by densitometry. In response to IFN-{gamma} stimulation, levels of both Jak1 and phospho-Jak2 were reduced in EHEC-infected cells to 21 and 49%, respectively, of those in uninfected controls.


Figure 5
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  		FIG. 5. EHEC infection disrupts Jak phosphorylation. T84 cells were infected with EHEC O157:H7 (MOI, 100:1) for 6 h at 37°C. Infected cells and uninfected cells were then stimulated with IFN-{gamma} (50 ng/ml) for 30 min at 37°C. Whole-cell protein extracts of T84 cells then were collected for Western blotting. Paired immunoblots were probed with either anti-Jak1 (1/1,000) (panel A, upper panel) and anti-ß-actin (1/5,000) primary antibodies (panel A, lower panel) or anti-Jak2 phosphospecific (pTyr10097/1008) (1:1,000) (panel B, upper panel) and anti-ß-actin (1:5,000) primary antibodies (panel B, lower panel). Positive bands were detected by scanning immunoblots using the Odyssey infrared imaging system. Integrative intensities of positively staining bands were calculated by densitometry with uninfected cells set to 100%. Both Jak1 (21% of control) and phospho-Jak2 (49%) bands were reduced in EHEC-infected cells relative to uninfected cells.

EHEC infection disrupts subcellular localization of IFN-{gamma} receptor domain 1. Mycobacterium avium infection decreases IFN-{gamma} receptor domain 1 and 2 expression in macrophages (20). Thus, we determined the expression and subcellular localization of the IFN-{gamma} receptor domain 1 using confocal microscopy. In an uninfected T84 monolayer, prominent IFN-{gamma} receptor domain 1 immunoreactivity occurred in the basal and mid-regions, but not the apical region of the cell (Fig. 6A, C, and E). However, T84 cells infected with EHEC O157:H7 strain CL-56 exhibited decreased staining in the basal and mid-regions, but increased staining in the apical region, compared with uninfected cells (Fig. 6B, D, and F). In the lateral view of a three-dimensional reconstruction from the T84 monolayer, IFN-{gamma} receptor domain 1 expression was seen throughout the uninfected cell (Fig. 6G), whereas the expression predominately localized to the apical aspect of EHEC-infected cell (Fig. 6H).


Figure 6
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  		FIG. 6. Subcellular localization of IFN-{gamma} receptor domain 1 (IFNGR1) with EHEC infection. T84 cells grown on glass coverslip slides were infected with EHEC O157:H7 strain CL-56 (MOI, 100:1, 6 h), fixed, permeabilized, blocked, and stained to visualize the IFNGR1 by confocal microscopy. Panels A (basal), C (mid), and E (apical), image sections from the basal, mid-, and apical regions of uninfected T84 cells, respectively. IFNGR1 expression mainly localizes to the basal and mid-regions of the T84 cells, and much less so to the apical region. Panels B (basal), D (mid), and F (apical), image sections from similar basal, mid-, and apical regions, respectively, of T84 cells that were infected with EHEC. IFNGR1 expression is decreased in the basal and mid-regions of the T84 cells, but increased in the apical region compared with uninfected cells. Panel G: Lateral view of a three-dimensional reconstruction from the uninfected T84 monolayer, where IFNGR1 is expressed throughout the cell. Panel H: Lateral view of a three-dimensional reconstruction from an infected T84 monolayer, where IFNGR1 expression is predominately localized at the apical aspect of the monolayer (a, apical; b, basal; n = 1 to 2). Approximate original magnification, x60.

Functional IFN-{gamma} receptor domain 1 localizes to caveolin-enriched plasma membrane microdomains. IFN-{gamma} receptor domain 1 localized to membrane microdomains has been suggested to contain the functional signal-transducing receptor for IFN-{gamma} (51). Fractionation of the Triton X-100 protein extract on a density gradient followed by immunoblot analysis demonstrated that a portion of the cellular pool of IFN-{gamma} receptor domain 1 was found in the caveolin-1-enriched membrane microdomain fraction of unstimulated T84 cells (fraction 3) (Fig. 7). Subsequent analysis of this membrane microdomain fraction showed that infection of T84 cells with both EHEC serotypes O157:H7 and O113:H21 caused decreased microdomain expression of IFN-{gamma} receptor domain 1 compared with uninfected cells (Fig. 7).


Figure 7
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  		FIG. 7. Disruption of IFN-{gamma} receptor domain 1 (IFNGR1) localization to caveolin-enriched membrane microdomains by EHEC infection. T84 cells were grown in 3-cm dishes, samples were pooled, and proteins were extracted in 1% Triton X-100, separated by density gradient ultracentrifugation, and analyzed by immunoblotting. Molecular weight markers are indicated on the left-hand side, and gradient fraction numbers along the top. IFNGR1 expression was found in the caveolin-enriched (panel E), membrane microdomain (fraction 3), in uninfected, unstimulated (panel A) T84 cells, and in cells treated with IFN-{gamma} (panel B). In T84 cells infected with EHEC O157:H7 (strain CL-56) (panel C) or EHEC O113:H21 (strain CL-15) (panel D) (MOI, 100:1, 6 h), IFNGR1 expression in fraction 3 was reduced irrespective of IFN-{gamma} stimulation (50 ng/ml).


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DISCUSSION
 
EHEC serotypes O157:H7 and O113:H21 both disrupt cytokine signal transduction in epithelial cells (4), but the mechanisms underlying this observation remain to be delineated. Herein, we show that bacteria physically separated from a T84 cell monolayer were unable to suppress Stat1 activation by IFN-{gamma}. In contrast, we provide mechanistic evidence that EHEC O157:H7 and O113:H21 both suppressed IFN-{gamma}-induced Stat1 activation when allowed to contact host cells. Reinforcing the notion that contact is necessary, conditioned medium from infected T84 cells, but not medium from T84 cells or bacterial culture supernatant alone, inhibited IFN-{gamma}-induced Stat1 activation. Furthermore, bacterial, but not host cell, protein synthesis was required to inhibit Stat1 activation, and inhibition was independent of protein tyrosine phosphatases, flagella, quorum sensing, or host cell proteasome activity. Lastly, with respect to host factors, EHEC O157:H7 infection disrupted the subcellular distribution of IFN-{gamma} receptor domain 1.

Adhesion of microbes to epithelial cells can be a critical step in the infection process (14). For example, Listeria monocytogenes binds specific host cell surface receptors before internalizing into epithelial cells (42). Adhesion is also critical in a different manner for bacteria that actively avoid internalization, such as EHEC O157:H7 (25). This attaching-and-effacing bacterium adheres to the cell surface and injects its own receptor known as translocated intimin receptor (Tir), or EspE, into the host cell via a type III secretion system (9, 21). Host membrane-based EspE then intimately binds to the bacterial outer membrane protein intimin, encoded by the eae gene on the LEE pathogenicity island. However, it is likely that the mechanism behind EHEC inhibition of IFN-{gamma}-induced Stat1 activation is distinct from the products of the LEE pathogenicity island and the attaching-effacing lesion, since a type III secretion mutant also suppressed IFN-{gamma}-induced Stat1 activation (4) and since O113:H21 is negative for the LEE marker eaeA (11). As contact between the bacteria and host cell is required, candidate bacterial factors include outer membrane proteins common to the two serotypes involved in adhesion, such as the chromosomally encoded long polar fimbriae (35, 55), a hypothesis that awaits testing.

Bacterial contact with eukaryotic cells induces the secretion of multiple proteins of both bacterial (6) and host cell (5) origin. We now describe, for the first time, that EHEC O157:H7 infection of T84 cells led to secretion of a factor(s) capable of suppressing Stat1 activation. This soluble factor is not dependent on quorum sensing for expression nor is it present in bacterial culture supernatants, providing further support for the contention that interaction between EHEC and T84 cells is necessary to suppress Stat1 activation. Furthermore, this factor may not be a peptide, since its ability to suppress Stat1 activation was resistant to treatment with proteinase K, trypsin, heat inactivation, and sodium orthovanadate. Indeed, bacteria can secrete nonpeptide factors that interact with host cells, such as the monocyte chemoattractant diethyl phthalate by Helicobacter pylori (27). However, since peptides can be heat inactivation resistant (16), and since protease treatment may leave behind an active, smaller peptide, the exact molecular identity of the factor(s) requires further determination.

We also found that the bacterial protein synthesis inhibitor chloramphenicol completely blocked the ability of EHEC O157:H7 to suppress IFN-{gamma}-induced Stat1 tyrosine phosphorylation in a time-dependent manner, whereas the inhibition of host cell protein synthesis using cycloheximide did not block the ability of EHEC O157:H7. Taken together, the present data suggest that the soluble mediator(s) inhibiting Stat1 signal transduction is shed or secreted from the bacteria in a protein synthesis-dependent manner.

Cytokine-induced signal transduction is negatively regulated by various complementary pathways, including induction of SOCS protein expression and phosphatase activity (45). For example, infection with L. monocytogenes (50) or treatment with Toll-like receptor ligands (8) inhibits IFN-{gamma} activation of Stat1 tyrosine phosphorylation in macrophages by inducing SOCS expression. Indeed, SOCS proteins may degrade Jak proteins through a proteasome-dependent mechanism to suppress cytokine signaling (45). Recently, respiratory syncytial virus-mediated suppression of Stat2 activity was shown to be dependent on the proteasome (37), and NF-{kappa}B activation has also been implicated in SOCS induction (32).

In this study, we provide multiple lines of evidence that induction of SOCS protein expression or proteasome activity by EHEC is likely not involved. First, two different EHEC O157:NM strains suppressed IFN-{gamma}-induced Stat1 activation, while flagellin did not, indicating a TLR5-independent mechanism. Next, inhibition of host cell protein synthesis by cycloheximide did not reverse the effects of EHEC-mediated Stat1 suppression, suggesting a SOCS-independent mechanism. Also, pharmacological inhibition of proteasome activity by MG-132 did not reverse EHEC-mediated Stat1 suppression. Additionally, because MG-132 can function as an NF-{kappa}B inhibitor, NF-{kappa}B activation by EHEC O157:H7 (2, 7) is likely not involved.

Since host cell phosphatases negatively regulate Stat1 tyrosine phosphorylation (18, 53), the possibility that EHEC O157:H7 and O113:H21 directly induced host cell phosphatase activity was also tested. Protein tyrosine phosphatases regulate IFN-{gamma}-Stat1 signaling by dephosphorylating activated Stat1 and Jak proteins (3, 41). Stat1 dephosphorylation was originally described at the host cell nucleus (18); however, the precise mechanism and host cell factor were not described.

Subsequently, a ubiquitous Stat1 protein tyrosine phosphatase was identified: T-cell protein tyrosine phosphatase (TcPTP) activity is present in cytoplasmic and nuclear extracts of hepatoma (Hep3B) cells, HeLa cells, and mouse embryonic fibroblasts (41, 53). Inhibition of TcPTP activity by orthovanadate results in Stat1 phosphorylation and Stat1 DNA binding in both cytoplasmic and nuclear extracts of IFN-{gamma}- and interleukin-6-stimulated cells (41).

In the current study, sodium orthovanadate treatment of infected T84 cells did not reverse the inhibitory effects of EHEC O157:H7 infection on Stat1 activation. Therefore, in accordance with the cycloheximide data, upregulation of a host cell protein tyrosine phosphatase likely is not responsible for the inhibitory effects of EHEC O157:H7 on activation of Stat1. Tyrosine phosphorylation of Stat1 did not occur when T84 cells were treated with sodium orthovanadate for 30 min or when the tissue culture cells were simultaneously treated with sodium orthovanadate and EHEC for 6 h. Taken together, these results argue against the involvement of a bacterial protein tyrosine phosphatase. In accordance with the proteinase K, trypsin, and heat inactivation data, the findings provide further evidence for the involvement of a nonpeptide bacterially derived factor in the inhibition of Stat1 tyrosine phosphorylation.

IFN-{gamma}-induced Stat1 signaling also can be regulated upstream of Stat1 proteins; for instance, at the level of Jak proteins, or further upstream by degradation, or down-regulation, of the IFN-{gamma} receptor. Jak1 and Jak2 are cytoplasmically linked to IFN-{gamma} receptor domains 1 and 2, respectively (1). Sodium orthovanadate specifically abolishes the interaction of Jak2 and TcPTP (46) and thereby allows for continued Jak phosphorylation and Stat protein activation (17). In the current study, Jak2 phosphorylation was reduced following EHEC infection relative to uninfected T84 cells. However, Jak2 phosphorylation was unchanged in EHEC-infected cells treated with sodium orthovanadate, suggesting that a host cell-derived protein tyrosine phosphatase may not be responsible for the observed inhibitory effects of EHEC infection on Stat1 activation. Instead, the results provide support for the concept that EHEC disruption of IFN-{gamma}-stimulated Stat1 activation occurs further upstream, at the level of the IFN-{gamma} receptor.

Recent evidence shows that multiple bacteria prevent activation of IFN-{gamma}-induced Stat1 activity, including Mycobacterium avium (20), Mycobacterium tuberculosis (54), L. monocytogenes (50), and EHEC O157:H7 and EHEC O113:H21 (4). Mechanistically, M. avium infection of macrophages leads to reduced mRNA and whole-cell protein expression of IFN-{gamma} receptor domains 1 and 2 and to decreased IFN-{gamma}-induced Stat1 tyrosine phosphorylation (20). Adenovirus also disrupts IFN-{gamma} signal transduction by decreasing IFN-{gamma} receptor domain 1 mRNA and cell surface protein expression (24). On the other hand, M. tuberculosis inhibits IFN-{gamma}-induced Stat1 activation in macrophages without affecting Stat1 tyrosine phosphorylation or DNA-binding ability (29, 54). Rather, M. tuberculosis decreases the association of activated Stat1 with transcriptional coactivator proteins (54) in a process dependent on bacterially induced interleukin-6 release from the macrophage (34). The present study provides evidence that EHEC O157:H7- and EHEC O113:H21-mediated suppression of Stat1 activation by IFN-{gamma} involves the production of a secreted mediator(s) and disrupted IFN-{gamma} receptor domain 1 expression in caveolin-enriched membrane microdomains.

Cholesterol and sphingolipid-enriched plasma membrane microdomains are thought to serve as platforms for signal transduction originating at the host cell surface (40). Protein receptors such as CC chemokine receptor 5 for macrophage inflammatory protein 1ß (36), gp130 for interleukin-6 signaling, and the IFN-{gamma} receptor domain 1 for IFN-{gamma} (40) all require localization to microdomains to retain functionality. It has recently become appreciated that microbes exploit membrane microdomains to their advantage (31, 38). For example, the cystic fibrosis transmembrane conductance regulator (CFTR) serves as a cell surface receptor for Pseudomonas aeruginosa on the apical aspect of epithelial cells, and CFTR must be localized to microdomains to allow bacterial internalization and activation of the proinflammatory NF-{kappa}B signal transduction cascade (30).

Here, we confirm that IFN-{gamma} receptor domain 1, which is important for binding of IFN-{gamma} (1), localizes to membrane microdomains of intestinal epithelial cells. Furthermore, EHEC O157:H7 and EHEC O113:H21 infection was shown to disrupt this localization to caveolin-enriched fractions. Whether conditioned medium mimics this effect or inhibition of bacterial protein synthesis using chloramphenicol reverses this effect remains to be determined. Nevertheless, these results indicate that bacteria can alter the distribution and function of signaling receptors in addition to exploiting the proteins present in membrane microdomains.

In summary, this study provides novel observations describing the mechanisms underlying EHEC suppression of cytokine signaling that are important for human defense against infection. Developing a better understanding of the pathogenesis of infection will help to guide new approaches to future therapeutic intervention strategies.


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ACKNOWLEDGMENTS
 
We thank Danny Aguilar at the Hospital for Sick Children Graphics Center for assistance preparing the figures.

N.J. was the recipient of a Cell Signals Trainee Award and an Open Fellowship award from the University of Toronto. P.J.M.C. was the recipient of a Canadian Institutes of Health Research (CIHR) Doctoral Student Award and a Cell Signals Trainee Award. D.M.M. is a CIHR scholar. P.M.S. is the recipient of a Canada Research Chair in Gastrointestinal Disease. This work was supported by operating grants from the CIHR.


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FOOTNOTES
 
* Corresponding author. Mailing address: Gastroenterology and Nutrition Room 8409, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8. Phone: (416) 813-7734. Fax: (416) 813-6531. E-mail: philip.sherman{at}sickkids.ca. Back

Editor: A. D. O'Brien

{dagger} N.J. and P.J.M.C. contributed equally to the planning and execution of this work and in the preparation of the manuscript. Back


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Infection and Immunity, March 2006, p. 1809-1818, Vol. 74, No. 3
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.3.1809-1818.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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