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J. Biol. Chem., Vol. 277, Issue 15, 13346-13353, April 12, 2002

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The Salmonella typhimurium Flagellar Basal Body Protein FliE Is Required for Flagellin Production and to Induce a Proinflammatory Response in Epithelial Cells^*

Katharine A. Reed^§, Michael E. Hobert^§¶, Claire E. Kolenda, Kara A. Sands, Michelle Rathman^**, Miriam O'Connor, Sean Lyons, Andrew T. Gewirtz, Philippe J. Sansonetti^**, and James L. Madara

From the  Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322, the  Department of Veterinary Pathobiology, Texas A & M University, College Station, Texas 77843, and ^** Unité de Pathogénie Microbienne Moléculaire, Paris Cédex 15, France

Received for publication, January 7, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

During apical colonization by Salmonella typhimurium, intestinal epithelial cells orchestrate a proinflammatory response that involves secretion of chemoattractants, predominantly interleukin-8, which coordinate neutrophil trans-epithelial migration at the site of infection. This host-pathogen interaction requires several S. typhimurium genes. To identify novel genes that participate in this pathogen-induced proinflammatory response, we created S. typhimurium Tn-10 transposon mutants and identified a single mutant with Tn-10 insertional inactivation within the fliE flagellar locus that was able to adhere to and invade intestinal epithelial cells normally but was unable to induce interleukin-8 secretion in host cells. The fliE-deficient mutant failed to secrete flagellin and lacked any surface assembly of flagellae. Unlike wild-type S. typhimurium, the fliE-deficient mutant did not activate the IB/NF-B signaling pathway or induce the coordinated trans-epithelial migration of isolated human neutrophils. Transcomplementation of the fliE-deficient mutant with a wild-type fliE-harboring plasmid restored all defects and produced a wild-type S. typhimurium phenotype. Furthermore, functional down-regulation of basolateral TLR5 completely inhibited the monolayers' ability to respond to both wild-type S. typhimurium and purified flagellin but had no affect on tumor necrosis factor -induced responses. We therefore conclude that S. typhimurium fliE is essential for flagellin secretion, flagellar assembly, and S. typhimurium-induced proinflammatory responses through basolateral TLR5 and is consistent with the emerging model of S. typhimurium flagellin-induced inflammation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The epithelial cells lining the gastrointestinal tract form a highly specialized barrier that separates two very distinct environments, thus maintaining the delicate balance between the gut lumen and the underlying tissue (1, 2). As part of its barrier function, the intestinal epithelium is able to detect surface attached enteric pathogens like Salmonella enterica, serovar Typhimurium (S. typhimurium), and orchestrate a proinflammatory response. This multifaceted response involves the rapid secretion of chemoattractants, the development of a chemotactic gradient in the surrounding subepithelial matrix (3-5), and the migration of neutrophils to the site of infection, ultimately triggering secretory diarrhea (6-11). The concurrent apical release of a soluble factor, designated PEEC (for pathogen-elicited epithelial chemoattractant), aids in directing the final polymorphonuclear leukocyte (PMN) movement across the epithelial tight junctions into the gut lumen (12). In addition to IL-8,¹ a variety of other chemokines are secreted in response to S. typhimurium attachment (13-15). Salmonella-induced secretion of IL-8 is known to require the activation of the nuclear transcription factor NF-B (16, 17). In quiescent cells, the NF-B heterodimer (p50/p65) is held in a stable, inactive, cytoplasmic complex with IB molecules until activated by an as yet undefined Salmonella-stimulated proinflammatory signaling cascade. The IB molecule is then phosphorylated and degraded, thus releasing the NF-B molecule to translocate to the nucleus, bind the IL-8 promoter, and induce transcription of the IL-8 gene (16).

The flagellae of S. typhimurium, like many other motile bacteria, are comprised of the basal body, the hook, and the filament, which is composed mainly of the protein FliC or FljB (dependent on phase variation) (18). The assembly of flagellae in Salmonella is similar to the secretion of virulence factors and requires the complex regulation of export machinery across both inner and outer bacterial cell membranes. Thus, it is not surprising that certain components of the secretion system utilized in flagellar biosynthesis are structurally and functionally homologous to components of the type III secretion system used for the export of virulence factors in both S. typhimurium (19-21) and Shigella (22). Additionally, the significant structural and functional similarities between these two secretory systems may indicate that flagellar export machinery is an additional mechanism for secretion of virulence factors from S. typhimurium (23) and other pathogens (24).

Recently, interest has been focused on Salmonella flagellin and its role in the induction of host proinflammatory responses. Studies have demonstrated that purified flagellin can induce proinflammatory mediators in epithelial cells (25) and must be translocated across the epithelial monolayer to the basolateral surface for these responses to occur (26). Additionally, flagellin has been shown to interact with the basolateral Toll-like receptor 5 (TLR5) on human intestinal epithelial cells (27) and can induce systemic responses in mice that require the cytosolic adaptor protein Myd88 (28).

Here we demonstrate that a Salmonella mutant in which the fliE gene has been disrupted by a Tn-10 transposon insertion (29) retains the ability to interact with intestinal epithelial cell apical membranes, but such interactions are nonproductive in terms of signaling epithelia to initiate proinflammatory signaling cascades. Disruption of fliE results in Salmonella mutants that are unable to secrete flagellin or assemble flagellae. Complementation of the fliE mutant strain with a plasmid containing wild-type fliE results in recovery of a wild-type Salmonella phenotype. We have also established for the first time that TLR5 is expressed exclusively on the basolateral membrane of Madin-Darby canine kidney cells and is functionally down-regulated by preincubation with purified flagellin. This TLR5 functional down-regulation results in monolayers that are insensitive to both flagellin and wild-type Salmonella colonization but does not affect TNF-stimulated IL-8 secretion. Taken together, we have demonstrated that the S. typhimurium fliE gene is required for secretion of flagellin, flagellar assembly, and the initiation of proinflammatory responses in host cells. Additionally, we have shown that these proinflammatory responses are initiated through flagellin-TLR5 interactions at the basolateral surface.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- Human model intestinal epithelial cells (T84, passages 50-65) were grown and maintained as confluent monolayers on collagen-coated permeable supports (30), with recent modifications (31). MDCK epithelial cells (passages 5-20) were also cultured and maintained as confluent monolayers in a 5% CO₂ humidified atmosphere at 37 °C. T84 monolayers were maintained in a 1:1 mixture of Dulbecco-Vogt modified Eagle's medium and Ham's F-12 medium supplemented with 15 mM HEPES buffer (pH 7.5) (Sigma); 14 mM NaHCO₃; 40 mg of penicillin, 8 mg of ampicillin, and 90 mg of streptomycin per ml; and 5% newborn calf serum. MDCK monolayers were maintained in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 14 mM NaHCO₃, 100 units/ml penicillin, and 100 µg/ml streptomycin. Monolayers of T84 and MDCK cells were grown on 96-well polycarbonate plates (MDCK) (Costar Corp., Cambridge, MA) or 0.33-cm² ring-supported polycarbonate filters (T84 and MDCK) of 0.4-µm pore size (Costar) and utilized 3-5 and 6-14 days postseeding, respectively. T84 monolayers received weekly and MDCK biweekly feedings postseeding. Cultured inverted monolayers of T84 cells, for PMN transmigration studies, utilized 5.0-µm pore size and were constructed as previously described (31, 32). All culture medium supplies were purchased from Invitrogen.

Bacterial Strains, Plasmids, and Growth Conditions-- All S. typhimurium and Escherichia coli strains were grown as previously described (3). Nonagitated, microaerophillic bacterial cultures were prepared by inoculating 40 ml of LB broth with 40 µl of a stationary phase culture, followed by overnight incubation at 37 °C (3). For construction and transfer of the Tn-10 transposon, we used the nonpathogenic E. coli strains DH5 and SM10 (pir). Transformations for sequence analysis utilized E. coli Top10-competent cells (Invitrogen Corp., Carlsbad, CA). Transcomplementation used the plasmid vector pTrc99A (Amersham Biosciences), harboring the wild-type fliE cDNA, and is designated PMM1001 (kind gift of R. M. Macnab, Yale University, New Haven, CT). Ampicillin (100 µg/ml), kanamycin (50 µg/ml), and naladixic acid (75 µg/ml) were added to bacterial culture medium when necessary. Purified flagellin was prepared as previously described (26).

Creation of a Tn-10 Transposon Bank-- A bank of random transposon mutants was created using a mini-Tn-10 derivative with a lacZ reporter in a 3306 S. typhimurium background. The Tn-10-lacZ derivative was constructed by ligating the lacZ gene from pGP704 into pBSL180, a suicide vector with resistance to kanamycin (Kan) (33). Using SM10pir, the Tn-10-lacZ transposon was conjugally transferred to 3306, and transconjugates were selected on naladixic acid and Kan media. A library of ~10,000 independent mutants was created. Random insertion of the transposon was verified by Southern hybridization using internal fragments as a probe. Recombinant DNA manipulations were carried out by standard methods (34).

Genetic Manipulations-- S. typhimurium mutants identified from initial screens were checked for single transposon insertion sites. To determine the site of transposon insertion, an EcoRI digest was performed, and the Kan fragment was ligated into pBluescript (Stratagene, La Jolla, CA), and the resulting constructs were transformed into XL1-Blue (Stratagene). The nucleotide sequence of DNA flanking the transposon was determined using universal primers that hybridize to the M13-reverse and T7 promoter sequences (Promega Corp., Madison, WI) or to 19- and 20-bp regions of the Kan resistance cassette (5'-GCTTCCATCCGAGTACGTG, 5'-GCTCGACGTTGTCACTGAAG). Sequencing chemistry was performed using an ABI PRIZM "Big Dye" terminator cycle sequencing ready reaction kit with amplitaque FS, and analysis was carried out on an ABI PRIZM 377 automated DNA sequencer (Applied Biosystems/PerkinElmer Life Sciences). Sequences were aligned using MacVector 5.0 software (International Biotechnologies Inc., New Haven, CT). Transcomplementation of KAR729 was carried out using plasmid pMM1001 containing wild-type fliE cDNA (a gift from R. M. Macnab). Competent KAR729 cells were transformed with plasmid DNA, and amp^r/kan^r colonies were selected for further investigation; this new strain was designated KAR729(pMM1001).

Induction of IL-8-- Confluent epithelial monolayers of T84 or MDCK cells were grown as stated and equilibrated with 1.0 ml of Hanks' balanced salt solution plus Ca²⁺, Mg²⁺, and 10 mM HEPES pH 7.4 (Sigma) (HBSS+) in the lower wells and 100 µl in the upper wells and incubated for 30-45 min at 37 °C. To infect monolayers, 10 µl of an S. typhimurium suspension (2 × 10⁹/ml (multiplicity of infection ~70)) was added to the apical well of the cultured monolayers and incubated for 1 h at 37 °C. Monolayers were then carefully washed of excess bacteria and placed in 300 µl of HBSS+ in the lower (basolateral) well and incubated at 37 °C. The basolateral medium was collected 4 h later, and the concentration of IL-8 was analyzed by ELISA. For cells in the 96-well format, the final 30 min was modified; 50 µl of a 1% Triton X-100 solution in 10 mM EDTA was added to the upper surface of the cells. Negative controls with HBSS+ alone and positive controls with TNF (100 ng/ml) added to the basolateral medium were employed in each assay.

IL-8 ELISA-- IL-8 was measured by a sandwich enzyme-linked immunosorbent assay (ELISA). For T84 cells, the assay performed was as previously described (3), with some modifications. For MDCK cells, a canine ELISA was developed. For both assays, 96-well plates were coated overnight at 4 °C with either goat anti-human IL-8 (8 µg/ml; R & D Systems, Minneapolis, MN) or mouse anti-rabbit IL-8 (1 µg/ml; a gift from R. Mrsny (Genentech)) antibodies. Secondary antibodies were rabbit anti-human IL-8 (8 µg/ml; Endogen, Woburn, MA) or rabbit anti-canine IL-8 (1:200; a gift from R. Mrsny). The detection antibody for both was goat anti-rabbit peroxidase conjugate (1:7500; Kirkegaard & Perry Laboratories, Gaithersburg, MD).

Swarm Plate Assay-- Semisolid agar (0.35%) was used to evaluate the motility of the following S. typhimurium strains: wild type (3306), Phop^c (CS022), fliE knock-out (KAR729), and transcomplemented fliE (KAR729(pMM1001)). Small wells were made in semisolid agar plates, loaded with 5 µl from a maximally invasive culture (described above) of each strain and incubated at 37 °C for 4 h. The motility of each Salmonella strain in the agar was evaluated by the diameter of the "halo" of colony growth produced.

S. typhimurium Adherence to and Invasion of Epithelial Monolayers-- Infection of T84 or MDCK epithelial monolayers was performed by the method described previously (3). Monolayers were placed in a 24-well tissue culture plate with 300 µl of HBSS+ added to the lower (basolateral) well and 100 µl of HBSS+ added to the upper (apical) well. After a 30-min equilibration, 10 µl of bacterial solution (~20 bacteria/epithelial cell) was added apically, and bacterial adherence and invasion were assessed after 1 h. Cell-associated bacteria represent bacteria adhered to and/or internalized into the monolayers and were released by incubation with 100 µl of 1% Triton X-100 (Sigma). Internalized bacteria were those obtained after lysis of the monolayer with 1% Triton X-100 following a 45-min incubation with gentamicin (50 µg/ml). For both cell-associated and internalized bacteria, 0.9 ml of Luria broth was added, mixed, and quantified by plating on MacConkey agar medium for colony-forming units. To determine the number of attached bacteria, the number of cell-internalized bacteria was subtracted from the number of cell-associated bacteria (since cell-associated bacteria include both attached and internalized bacteria).

PMN Transepithelial Migration Assay-- The physiologically relevant (basolateral to apical) transepithelial migration of PMN (neutrophils) using cell culture inserts of inverted T84 monolayers has previously been detailed (3, 32). Human PMN were isolated from normal volunteers as described (32, 35). Inverted T84 cell monolayers were washed in HBSS+ to remove residual serum components. S. typhimurium was washed twice in HBSS+ and resuspended at a final concentration of ~5 × 10⁹ bacteria/ml. 100-µl bacterial aliquots (~5 × 10⁸ bacteria) were microcentrifuged at 14,000 × g for 3 min and resuspended in a final volume of 25 µl. Inverted monolayers were placed in a humidified chamber with the apical membrane oriented upward. The bacterial suspension was added to the apical surface and incubated for 1 h at 37 °C. Nonadherent bacteria were removed by washing three times in HBSS+. The monolayers were then transferred back into the 24-well tissue culture tray containing 300 µl of HBSS+ in the lower reservoir (i.e. the apical membrane now colonized with Salmonellae), and 160 µl was added to the upper reservoir (i.e. the basolateral interface). For simplicity, the reservoir will be referred to by the epithelial membrane domain with which it interfaces. (i.e. apical or basolateral). To the basolateral chamber, 40 µl (~10⁶) of isolated PMNs were added to each monolayer and incubated for 110 min at 37 °C. The addition of 1 µM N-formylmethionylleucyl phenylalanine to the apical reservoir served as a positive control. Transmigration was quantified by assaying for the PMN azurophilic granule marker myeloperoxidase as described previously (32, 36). After each transmigration assay, nonadherent PMNs were washed from the surface of the monolayer, and PMN cell equivalents, estimated from a standard curve, were assessed as the number of PMNs associated with the monolayer and the number that had completely traversed the monolayer (i.e. into the basolateral reservoir).

Cell Surface Biotinylation-- Filter-grown cells were rinsed twice in ice-cold PBS supplemented with 0.1 mM CaCl₂ and 1 mM MgCl₂ (PBS-CM) and incubated for 30 min on ice with 1 mg/ml sulfo-N-hydroxysuccinimide-biotin (Pierce) in PBS-CM added to either the apical or basolateral compartments. Biotinylation was quenched by incubating monolayers for 5 min in 50 mM NH₄Cl in PBS-CM, washed three times in PBS, and lysed for 10 min on ice in a solution containing 1% Triton X-100, 20 mM Tris, pH 8.0, 50 mM NaCl, 5 mM EDTA, 0.2% bovine serum albumin supplemented with protease inhibitors. Biotinylated proteins were isolated by incubation with streptavidin-agarose beads (Pierce) for 16 h at 4 °C. Proteins bound to agarose beads were solubilized in protein loading buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol), separated by SDS-PAGE, and transferred to nitrocellulose membrane for immunoblotting.

Western Blot Analysis-- Total cell proteins were separated by SDS-PAGE (12%), transferred to nitrocellulose (Bio-Rad; 0.45-m membrane) and incubated in the presence of anti-IB mAb (1:2000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by a peroxidase-conjugated goat anti-rabbit antibody (1:2000; Amersham Biosciences). Bacterial flagellin was detected in bacterial lysates using anti-E. coli flagellin mAb (15D8) (1:1000; Igen International, Gaithersburg, MD), followed by goat anti-mouse peroxidase-conjugated antibody (1:2000; Amersham Biosciences). TLR5 was detected with anti-TLR5 polyclonal Ab (1:1000; Santa Cruz Biotechnology) followed by a peroxidase-conjugated goat anti-rabbit antibody (1:2000; Amersham Biosciences). Reactive bands were visualized by enhanced chemiluminescence using a kit (Amersham Biosciences) according to the manufacturer's instructions.

Confocal Microscopy-- T84 and MDCK monolayers were washed in ice-cold PBS and fixed in 3.7% paraformaldehyde in PBS (Electron Microscopy Sciences, Ft. Washington, PA) for 10 min. Monolayers were washed and permeabilized for 10 min in 0.2% Triton X-100. Actin filaments were stained with rhodamine-conjugated phalloidin for 45 min at 37 °C and rinsed three times in PBS. Monolayers attached to membranes were excised from the inserts and mounted cell-side up on a glass slide. The membrane was covered with SlowFade reagent (Molecular Probes, Inc., Eugene, OR), and a coverslip and edges were sealed to prevent drying. Specimens were examined with a Zeiss LSM410 scanning laser confocal microscope using the 488/568-nm wavelength lines of an argon-krypton laser. The cell monolayer was optically sectioned every 0.5 µm. Image resolution using a Zeiss ×100 Neofluor objective and Zeiss LSM software was 512 × 512 pixels.

Transmission Electron Microscopy-- Bacterial pellets were fixed in 4% gluteraldehyde solution over night at 4 °C for subsequent processing. Transmission electron microscopy processing was as previously described (3).

Statistical Analysis-- Paired Student's t test analysis was carried out on data using SSPSS software to assess statistical significance.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tn-10 Insertional Inactivation of Salmonella fliE Prevents Induction of IL-8 Secretion-- Approximately 1000 Salmonella Tn-10 mutants were screened for their ability to induce the secretion of IL-8 using MDCK cells in a 96-well format. One S. typhimurium Tn-10 mutant (designated KAR729) was identified in which the ability to induce IL-8 secretion in epithelial cells was consistently and significantly reduced (p < 0.001) (Fig. 1A). Using filter-grown monolayers, we observed a complete lack of IL-8 secretion in polarized monolayers infected with KAR729 that was identical to untreated controls in both MDCK (Fig. 1B) and T84 cells (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   S. typhimurium Tn-10 mutant KAR729 lacks the ability to induce IL-8 secretion. A, a small sample of the ~1000 Tn-10 mutants screened for their ability to induce IL-8 secretion using a canine IL-8-specific ELISA and MDCK cells in a 96-well format. KAR729 is identified. B, WT (3306), PhoPc (CSO22), and fliE mutant (KAR729) S. typhimurium strains were assessed for their ability to induce IL-8 secretion from filter-grown MDCK cell monolayers. Basolateral supernatants collected from apically infected filter-grown MDCK cell monolayers were analyzed by ELISA. Control MDCK monolayers were incubated with buffer alone, whereas positive controls were incubated with TNF added to the basolateral buffer. Data are presented as the mean ± S.D. of assays performed in triplicate. C, schematic diagram of Tn-10 transposon disruption of the fliE gene of S. typhimurium KAR729. S. typhimurium genomic DNA EcoRI fragments were cloned into pBluescript and sequenced using primers to T7 and M13 as well as primers to regions of the KanR cassette adjacent to the insertion sequences (IS10) of the Tn-10 transposon. Allocation of the genes upstream and downstream of fliE is based on sequence analysis of the 5' and 3' DNA regions.

To determine the insertion site of the Tn-10 transposon in the KAR729 mutant, EcoRI-digested genomic DNA fragments were inserted into pBluescript and sequenced using oligonucleotide primers to the T7 and M13 promoters as well as primers complementary to 19- and 20-bp regions within the kan^r sequence of the transposon (see "Experimental Procedures"). Sequence analysis revealed that the Tn-10 transposon had inserted within the S. typhimurium flagellar fliE locus (Fig. 1C) that is positioned at the end of the flagellar IIIb region of the S. typhimurium chromosome, at centisome 42 (37).

KAR729 Does Not Secrete Flagellin and Lacks Flagellae but Adheres to and Invades Epithelial Cells Normally-- Since the fliE gene, disrupted by the Tn-10 insertion, encodes a protein localized to the basal body of flagellae (38, 39), we examined the state of bacterial flagellae in KAR729. Currently, the understanding of the underlying mechanisms of flagellin protein secretion is incomplete, and the role FliE protein may play in this process is not well understood. We next assessed whether flagellin was secreted from the KAR729 mutant. Flagellin (FliC) is normally found in abundance in S. typhimurium supernatants and is easily detected by immunoblot. As shown in Fig. 2A, wild-type S. typhimurium 3306, the invasion-deficient mutant PhoP^c, and even E. coli (strain F18) secrete significant amounts of flagellin into the bacterial supernatant. However, both KAR729 and fliC/fljB (a flagellin-deficient mutant) do not secrete flagellin into the supernatant (Fig. 2A). Wild-type S. typhimurium 3306 is known to possess 8-10 flagellae per bacterium that are readily visualized by electron microscopy (Fig. 2B); however, KAR729 completely lacks flagellae (Fig. 2B). These findings suggest that fliE is not only required for flagellin secretion but also for flagellar assembly and are in complete agreement with other recent findings that demonstrate that completion of basal body assembly is necessary for flagellation (23, 39).

View larger version (55K): [in this window] [in a new window]   Fig. 2.   S. typhimurium Tn-10 mutant KAR729 does not secrete flagellin (FliC), lacks flagellae, and is nonmotile. A, bacterial supernatants collected from S. typhimurium 3306 (wild type), PhoPc mutant, E. coli F-18 (a flagellated E. coli strain), KAR729, and fliC/fljB (a flagellin-deficient S. typhimurium mutant) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with an anti-flagellin mAb. The 55-60-kDa flagellin band seen in all other supernatants was absent in KAR729 and fliC/fljB supernatants. Equal amounts of supernatant from equivalent numbers of bacteria were added to each lane. B, electron micrographs of S. typhimurium strains taken at ×40,000 magnification. Normal flagellae are indicated by arrows. C, equivalent numbers of bacteria were added to each well in semisolid agar and incubated for 4 h at 37 °C. Bacterial mobility was assessed by its ability to move, or swarm, through the agar, producing a large "halo."

To determine whether the biochemical and morphological lack of flagellin observed in KAR729 had an effect on bacterial motility, we performed a motility assay. Semisolid agar provides sufficient physical resistance to nonflagellated bacterial strains to prevent their movement and spread through the medium but allows free movement of flagellated strains. As demonstrated in Fig. 2C, wild-type 3306 was able to easily move through the semisolid agar and form a large colony; however, the less flagellated E. coli and PhoP^c formed only small colonies. Not surprisingly, we observed a complete lack of movement by KAR729 (Fig. 2C). Taken together, these data support the idea that the Tn-10 disruption of fliE in KAR729 produces a nonflagellated Salmonella strain.

Given the complete lack of flagellae in KAR729, we next assessed whether the inability to induce IL-8 secretion from host epithelial cells was a result of an inability to interact with the apical cell surface of host epithelium. We have previously shown that adherence, but not invasion, is required for productive interactions between Salmonella and host epithelium (40). Surprisingly, the ability of KAR729 to adhere to and invade both MDCK and T84 cell monolayers was virtually identical to wild-type 3306 (Fig. 3, A and B). As expected, the noninvasive mutant PhoP^c showed both reduced adherence and very low levels of invasion (Fig. 3, A and B). These data demonstrate that the failure of KAR729 to induce IL-8 secretion from epithelial monolayers is not a result of an inability to properly interact with the apical membrane.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   S. typhimurium Tn-10 mutant KAR729 adheres to and invades the apical surface of T84 and MDCK monolayers inducing localized actin cytoskeletal remodeling. Filter-grown T84 and MDCK cell monolayers were incubated for 1 h at 37 °C with equal amounts of maximally invasive bacterial cultures added to the apical chamber. The number of adherent (A) and invasive (B) bacteria was then determined for each bacterial strain (see "Experimental Procedures"). The noninvasive S. typhimurium strain PhoPc was used as a negative control. Data are presented as the mean ± S.D. of triplicate samples. C, filter-grown monolayers were infected apically for 30 min at 37 °C with WT (3306) or KAR729. Cells were fixed, permeabilized, and stained with rhodamine phalloidin and anti-Salmonella mAb followed by a fluorescein isothiocyanate-conjugated secondary Ab (see "Experimental Procedures"). Intact cell monolayers were optically sectioned every 0.5 µm. Horizontal (x-y) sections were taken in the plane of the apical (Ap) membrane (lower panels). Digitally compiled vertical (x-z) sections perpendicular to the plane of the apical membrane are shown directly above x-y sections. The arrows indicate sites of bacterial invasion and actin remodeling.

KAR729 Induces Rapid Host Cell Cytoskeletal Remodeling but Fails to Induce Early Events Required for Nuclear Signaling-- During the initial stages of bacterial-host interactions, Salmonella uses a type III secretory apparatus to inject bacterial effector proteins into the host cytoplasm (for a review, see Ref. 21), many of which have been shown to reorganize actin filaments just beneath the sight of bacterial attachment (41, 42). Since KAR729 physically interacts with the apical surface of host cells but fails to initiate pro-inflammatory responses, we sought to determine whether KAR729 could induce the rapid actin cytoskeletal remodeling. In monolayers labeled with both rhodamine phalloidin and an anti-Salmonella antibody, confocal microscopy of infected monolayers revealed that the KAR729 mutant not only adhered to the apical surface of epithelial cells but also induced localized alterations in the host cell cytoskeleton similar to the wild-type 3306 (Fig. 3C).

Following the rapid cytoskeletal remodeling, early signaling events result in activation of the IB-kinase complex, followed by nuclear translocation of the transcription factor NF-B and nuclear signaling. This process involves phosphorylation of the inhibitory molecule IB by IB-kinase, followed by ubiquitination and subsequent degradation in a proteasome. Degradation of IB provides an excellent indicator of activation of the NF-B signaling pathway. As shown in Fig. 4, incubation of monolayers with basolateral TNF or apical wild-type 3306 led to an almost complete degradation of IB, as detected by immunoblot. However, monolayers that were incubated with either PhoP^c, KAR729, or the nonflagellated fliC/fljB, had levels of IB comparable with that seen in untreated control monolayers (Fig. 4). Taken together, these results demonstrate that while KAR729 has retained the ability to induce rapid localized cytoskeletal remodeling in the host cell, it has lost the ability to stimulate signaling pathways that lead to IB degradation and the subsequent NF-B-mediated cytokine expression.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   S. typhimurium Tn-10 mutant KAR729 does not induce IB degradation. MDCK monolayers were incubated for 1 h at 37 °C with equal amounts of maximally invasive bacteria added to the apical chamber. Control monolayers were incubated with buffer alone or with basolateral TNF (100 ng/ml) as a positive control. Equal amounts of total cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-IB mAb followed by a secondary horseradish peroxidase-conjugated Ab. Bands were visualized using ECL (Amersham Biosciences).

KAR729 Does Not Induce Spontaneous Transepithelial Migration of PMN-- The proinflammatory program in epithelia, which is triggered by apical membrane attachment of wild-type S. typhimurium, orchestrates PMN movement toward and across the epithelium (9, 40, 43). We next sought to establish whether PMN transepithelial migration is induced by apical colonization of T84 monolayers with KAR729. Inverted confluent monolayers were used to analyze directed neutrophil migration in the physiologically relevant basolateral to apical direction. In this assay, the apical surface of T84 cell monolayers was incubated for 1 h in the presence of bacteria followed by washing to remove nonadherent bacteria. We then assessed the ability of the infected epithelial monolayers to orchestrate the PMN trans-epithelial migration by adding human PMN to the basolateral compartment. As shown in Fig. 5, we found that not only KAR729, but also fliC/fljB (the control nonflagellated strain) had a dramatically reduced (~90%) ability to direct the movement of PMN to the apical aspect of the monolayers when compared with wild-type 3306.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   S. typhimurium Tn-10 mutant KAR729 does not induce transmigration of PMN across epithelial monolayers. WT (3306), PhoPc, KAR729, or fliC/fljB S. typhimurium strains were assessed for their ability to promote PMN transmigration in the basolateral to apical direction. Monolayers of human T84 cells were incubated for 45 min with apically applied bacteria, followed by the addition of human PMN to the basolateral chamber. After a 110-min incubation, PMN transmigration was quantified by assaying for the PMN azurophilic granule marker myeloperoxidase in the apical chamber. The PMN chemoattractant N-formylmethionylleucyl phenylalanine (fMLP) (0.1 µM) was used as a positive control. Data are presented as the mean ± S.D. of triplicate samples.

Trans-complementation of KAR729 with Wild-type fliE Restores a Wild-type S. typhimurium Phenotype-- To determine whether the observed inability of KAR729 to induce signal transduction events necessary to promote IL-8 secretion is directly a result of fliE inactivation, we have complemented the defective fliE in KAR729 with wild-type fliE on an expression plasmid. The inducible expression plasmid pTrc99A carrying the wild-type fliE gene (pM1001) was introduced into KAR729 S. typhimurium cells by transformation. All of the resulting colonies were resistant to ampicillin and had normal colony morphology indistinguishable from wild-type 3306. The addition of wild-type fliE into the KAR729 mutant resulted in the inducible expression of flagellin as detected by immunoblot of bacterial protein extracts and Transmission Electron Microscopy (Fig. 6, A and B). The expression of wild-type fliE in KAR729 restored the ability of this strain to move through semisolid agar in a manner identical to wild-type 3306 (Fig. 6C). More importantly, expression of wild-type fliE reestablished the ability of the KAR729 mutant to induce IB degradation (Fig. 6D), provoke IL-8 secretion (Fig. 6E), and stimulate PMN transepithelial migration from basolateral to apical surface across T84 cell monolayers (Fig. 6F). Taken together, these data demonstrate that the only defect in KAR729 was the lack of FliE, and restoration of a wild-type S. typhimurium phenotype was achieved by reintroduction of the wild-type fliE gene.

View larger version (56K): [in this window] [in a new window]   Fig. 6.   Transcomplementation of KAR729 with wild-type fliE restores the wild-type S. typhimurium phenotype. KAR729 was transformed with a plasmid containing wild-type fliE (pM1001), and drug-resistant clones are screened. A, bacterial supernatants were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-flagellin mAb. B, electron micrographs of wild-type 3306 and KAR729(pM1001); the arrows indicate flagellae. C, bacterial motility in semisolid agar. D, IB immunoblot. E, S. typhimurium-induced IL-8 secretion; F, PMN transepithelial migration. Graphical data are presented as the mean ± S.D. of triplicate samples.

Functional Down-regulation of Basolaterally Expressed Toll-like Receptor 5 (TLR5) Inhibits Salmonella- and Flagellin-induced Epithelial Secretion of IL-8-- It is becoming increasingly clear that Salmonella-induced epithelial proinflammatory responses are mediated by soluble flagellin through interaction with basolateral TLR5 (25-28). Since our model epithelia are unresponsive to Salmonella mutants lacking flagellin secretion (KAR729, fliC/fljB), we sought to determine whether MDCK cells express TLR5 in a polarized manner and, second, whether functional down-regulation of TLR5 would inhibit the monolayers ability to respond to Salmonella. Using cell surface-specific biotinylation, MDCK cell monolayers were incubated with sulfo-N-hydroxysuccinimide-biotin (Pierce) applied to either the apical or basolateral chambers, quenched, lysed, and affinity-purified with streptavidin-agarose beads (Pierce). The isolated proteins were separated by SDS-PAGE and immunoblotted with anti-TLR5 antibody (Santa Cruz Biotechnology). MDCK monolayers expressed TLR5 predominantly (>95%) at the basolateral membrane as a ~100-kDa doublet that is a result of differential glycosylation (Fig. 7A). Thus, it is clear that MDCK monolayers have the same polarized TLR5 expression as has been previously reported for human T84 cell monolayers (27). We next sought to determine whether the epithelial proinflammatory response to Salmonella or purified flagellin could be inhibited by functional down-regulation of basolateral TLR5. This was achieved by preincubation of monolayers for 24 h with medium containing purified flagellin in the basolateral compartment. The monolayers were washed extensively and then incubated for 5 h with either wild-type Salmonella (3306) applied apically or purified flagellin in the basolateral chamber. The basolateral supernatants were collected, and secreted IL-8 was quantified by a canine-specific ELISA. Control monolayers that were not preincubated with basolateral flagellin responded normally to wild-type Salmonella, basolateral flagellin, and TNF (a positive control) (Fig. 7B). However, monolayers that had been preincubated with basolateral flagellin did not secrete IL-8 in response to either wild-type Salmonella or basolateral flagellin, while the response of the monolayers to TNF was unaffected (Fig. 7B). Taken together, these results dramatically demonstrate three things: first, MDCK monolayers are equipped with basolateral TLR5 exquisitely poised to respond to Salmonella colonization in a polarized manner; second, that functional down-regulation of TLR5 completely blocks the ability of the epithelial monolayer to mount a proinflammatory response to Salmonella; and, finally, that the flagellin/TLR5 interaction is the sole mediator of the epithelial proinflammatory response to Salmonella.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   TLR5 is required for S. typhimurium- and flagellin-induced IL-8 secretion. A, filter-grown MDCK monolayers were biotinylated at either the apical or basolateral surfaces, and biotinylated proteins were isolated by affinity chromatography. Biotinylated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-TLR5 antibody. B, filter-grown MDCK monolayers were incubated for 24 h at 37 °C in the presence or absence of purified flagellin (100 ng/ml) added to the basolateral medium. Cells were then washed and incubated for 5 h in the presence or absence of TNF (100 ng/ml) or purified flagellin (100 ng/ml) added basolaterally or apical WT Salmonella (3306). Basolateral supernatants were analyzed for IL-8 using ELISA. Graphical data are presented as the mean ± S.D. of triplicate samples from three separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The goal of this study was to identify novel S. typhimurium genes involved in the pathogen-induced proinflammatory response. This was achieved by creating random Tn-10 transposon insertion mutants, which were then screened for a reduction in their ability to induce epithelial IL-8 secretion. A single mutant was identified containing an insertional inactivation of the Salmonella fliE gene that not only completely lacked the ability to induce epithelial IL-8 secretion but also lacked flagellae and flagellin secretion. Using genetic, biochemical, and cell biological methods, we have demonstrated the role of fliE in the secretion and assembly of flagellin into flagellar structures and verified its importance in the inflammatory process.

Disruption of the S. typhimurium fliE gene prevents the secretion of flagellin and flagellar assembly. The product of the fliE gene, a protein of ~11 kDa, has been previously localized to the basal body of the flagellum (23, 44). FliE has also recently been shown to interact with the flagellar hook protein FlgB, a component of the basal body, and is thought to be required for the export of flagellar hook and rod components (39, 40, 44, 45). Although the precise role of FliE in flagellar assembly is not well understood, our results are in agreement with data from Komoriya et al. (23), who previously observed that any defect in a flagellar rod protein resulted in the absence of flagellae. We have also found that while this lack of flagellar assembly prevents bacterial locomotion, it does not affect bacterial adherence or invasion of epithelial cells. Presumably, the Salmonella type III effector proteins necessary for invasion are secreted normally, since rapid, localized actin remodeling and bacterial invasion were observed with the fliE-deficient mutant. This is consistent with recent observations that nonflagellated S. typhimurium mutants secrete higher levels of type III virulence factors and suggests a putative relationship between the two secretion systems (23).

Orchestration of PMN migration across an epithelial monolayer in response to S. typhimurium colonization requires recognition of the bacterial invader in a milieu of prokaryotic flora before secretion of basolateral IL-8 and apical PEEC can occur. Previously accepted models of Salmonella-induced proinflammatory responses developed from studies in nonpolarized cells relied on Salmonellae secretion of type III effector proteins to induce nuclear events necessary for proinflammatory responses (46, 47). However, two recent studies have demonstrated a definite link between flagellin secretion by Salmonella and induction of proinflammatory responses in epithelial cells (25, 26). Of late, the role of flagellin (FliC) in stimulating the proinflammatory response through TLR5 receptors has been reported (27, 28). We have shown that Salmonella focally infect epithelial cells in polarized monolayers, yet even those epithelial cells that are not surface-colonized uniformly signal through nuclear NF-B (48). This result suggested (and it was subsequently shown) that epithelial transcytosis of a proinflammatory factor that diffuses laterally has an affect on all cells in the monolayer, thus explaining the discrepancy between focal infection and the uniform epithelial response. Additionally, flagellin by itself is able to induce epithelial proinflammatory activation when added basolaterally but not apically (26).

Not surprisingly, we have found that fliE-dependent secretion of flagellin is necessary to induce proinflammatory activation of the IB/NF-B signaling pathway, IL-8 secretion, and PMN trans-epithelial migration. This fliE-deficient phenotype can be completely rescued by trans-complementation with the wild-type fliE gene. This is the first report of the regulatory role of FliE expression on the secretion of flagellin. Our results complement previous work demonstrating the role of flagellin in the innate immune response and would seem to be in contrast to the previous notion that Salmonella type III effector proteins translocated into the cytoplasm directly activate proinflammatory nuclear signaling events (46). However, the two ideas may not be mutually exclusive; secreted flagellin can activate proinflammatory signaling through the basolateral TLR5, and type III effectors may provide the stimulus by which flagellin is transcytosed across the epithelium. This idea is in complete agreement with a recent study that demonstrates that translocation of flagellin is critical for eliciting proinflammatory responses (26). However, flagellin is also synthesized and secreted by many nonpathogenic bacteria that are capable of colonizing the apical surface of epithelial monolayers, yet these bacteria do not induce a proinflammatory response. In aggregate, these findings paired with other current findings suggest that a critical difference between pathogenic and nonpathogenic bacteria is the ability to induce transcytosis of flagellin. In turn, this suggests that bacterial virulence factors, probably provided by injection via the type III secretory apparatus, may play an important role in usurping the transcytotic pathway as a means of delivering a critical proinflammatory factor (flagellin) to the responsive basolateral domain.

In summary, we have established that expression of the S. typhimurium fliE gene is necessary for bacterial secretion of flagellin and the assembly of functional flagellae. Additionally, using physiologically relevant epithelial model systems, we have demonstrated that S. typhimurium fliE-regulated secretion of flagellin is necessary to stimulate epithelial proinflammatory pathways that lead to IL-8 secretion and ultimately direct PMN trans-epithelial migration. While FliE is not a direct mediator of this immune inflammatory response, it does play a pivotal role in the secretion of flagellin. We have also demonstrated that TLR5 signaling can be functionally down-regulated by preincubation with purified flagellin. Further characterization of the mechanisms by which flagellin is translocated across the intestinal epithelium and how it interacts with TLR5 to induce proinflammatory signals may provide a better understanding of the aberrant signaling that occurs in chronic inflammatory diseases of the intestine.

	ACKNOWLEDGEMENTS

We especially thank Denice Esterly and Millie Pochet for technical assistance, Bob Santianni for assistance with Transmission Electron Microscopy and Randall J. Mrsny (Genentech Inc.) for supplying canine IL-8 antibodies.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DK-10085 (to M. E. H.) and DK-35932 and DK-47622 (to J. L. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ These authors contributed equally to this work.

^¶ To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, Emory University, 615 Michael St., Rm. 125 Whitehead Research Bldg., Atlanta, GA 30322. Tel.: 404-712-2817; Fax: 404-727-8538; E-mail: mhobert@emory.edu.

Published, JBC Papers in Press, January 30, 2002, DOI 10.1074/jbc.M200149200

	ABBREVIATIONS

The abbreviations used are: IL, interleukin; MDCK, Madin-Darby canine kidney; TNF, tumor necrosis factor; Kan, kanamycin; ELISA, enzyme-linked immunosorbent assay; PMN, polymorphonuclear leukocyte(s); PBS, phosphate-buffered saline; WT, wild type.

	REFERENCES

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