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Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan
Correspondence
Kei-ichi Uchiya
kuchiya{at}ccmfs.meijo-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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B, nuclear factor B; PAMP, pathogen-associated molecular pattern; SOCS, suppressor of cytokine signalling; SPI-2, Salmonella pathogenicity island 2; TLR, Toll-like receptor; TTSS, type III secretion system |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) is an essential contributor to macrophage activation and promotes the effective killing of pathogens that can survive within macrophages (Bhardwaj et al., 1986
; Green et al., 1990; Suzuki et al., 1988; van Dissel et al., 1987). On the other hand, macrophage functions can be blocked by interleukin-4 (IL-4), IL-10 and transforming growth factor-β, as well as by prostaglandin E2 (Bogdan & Nathan, 1993; Kunkel et al., 1988; O'Farrell et al., 1998; Strassmann et al., 1994). Pathogen-associated molecular patterns (PAMPs), which are components of bacteria and viruses, are able to induce the expression of mediators by several types of cells, including macrophages, and influence the host immune system (Janeway & Medzhitov, 2002). PAMPs include cell wall components derived from Gram-positive bacteria, LPS from Gram-negative bacteria, lipoteichoic acid, and flagellum, all of which are recognized by Toll-like receptors (TLRs). Specifically, bacterial cell wall components and lipoteichoic acid are recognized by TLR2 (Schwandner et al., 1999), and LPS and flagellin bind TLR4 and TLR5, respectively (Hayashi et al., 2001; Poltorak et al., 1998). Ligand binding by TLRs leads to the induction of immune and inflammatory genes (Akira & Takeda, 2004).
Salmonellae are Gram-negative bacteria that cause a variety of disease syndromes in humans and animals. Salmonellae invade and destroy specialized epithelial cells in the host intestine, and migrate to the mesenteric lymph nodes, where they encounter, and subsequently survive and replicate within macrophages. Specific virulence factors encoded within the Salmonella pathogenicity island (SPI) are required at various stages of Salmonella infection (Groisman et al., 1999). Salmonella enterica serovar Typhimurium, which causes gastroenteritis in humans and a systemic disease in mice similar to human typhoid fever, harbours two kinds of type III protein export systems: one for flagellar proteins as described above, and one for virulence factors. Several genes from the two systems share sequences in common (Aizawa, 2001; Macnab, 2004). Some important virulence factors are directly delivered into the host environment by two different type III secretion systems (TTSSs) encoded on SPI-1 and SPI-2. The SPI-1 TTSS mediates bacterial entry into nonphagocytic cells (Galan, 2001), and the SPI-2 TTSS is required for survival and replication in the intracellular environment of host cells and for systemic infection in mice (Hensel et al., 1998; Ochman et al., 1996; Shea et al., 1996).
SpiC is a virulence factor encoded within SPI-2. Previous studies have shown that a strain carrying a mutation in the spiC gene is unable to survive within macrophages and has greatly reduced virulence in mice. The SpiC protein is necessary for inhibiting the fusion of Salmonella-containing phagosomes with endosomal and lysosomal compartments (Uchiya et al., 1999). Moreover, this protein is translocated by the SPI-2 TTSS to the cytosol of macrophages, where it interacts with host proteins such as TassC (Lee et al., 2002) and Hook3 (Shotland et al., 2003) to alter intracellular trafficking. On the other hand, several investigators have reported that SpiC is required for the translocation of SPI-2 effector proteins into target cells by interacting with SsaM, a SPI-2-encoded protein (Freeman et al., 2002; Yu et al., 2002, 2004). In addition, our recent studies have shown that SpiC is involved in Salmonella-induced activation of the signal transduction pathways in macrophages, leading to not only the production of IL-10 and prostaglandin E2 but also the expression of suppressor of cytokine signalling (SOCS)-3, which is involved in the inhibition of IL-6 and IFN- signalling (Uchiya et al., 2004; Uchiya & Nikai, 2004, 2005). As described above, the production of these mediators is thought to have an important role in the intracellular survival of Salmonella and its escape from the host defence system via modification of macrophage function.
In the present study, we investigated the mechanism by which SpiC mediates the activation of signal transduction pathways in macrophages following Salmonella infection. We found that SpiC promotes the expression of FliC at the transcriptional level. This expression of FliC activates mitogen-activated protein kinase (MAPK) signalling pathways, which play a significant role in the SPI-2-dependent induction of SOCS-3 expression by Salmonella infection.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and Uchiya et al. (1999), respectively. The deletion mutants of fliC and fljB gene were constructed using the Red recombination system (Datsenko & Wanner, 2000). To delete the fliC gene, a kanamycin-resistance gene flanked by FLP recognition target sites from plasmid pKD4 was amplified by PCR using primers with regions homologous to the fliC gene (5'-GATCATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACGTGTAGGCTGGAGCTGCTTC-3' and 5'-ATCAATCGCCGGATTAACGCAGTAAAGAGAGGACGTTTTGCGGAACATATGAATATCCTCCTTAGT-3'). Kanamycin-resistant strains were obtained by transforming the PCR products into strain 14028s harbouring the 
Red recombinase on plasmid pKD46. Disruption of the fliC gene was confirmed by PCR using the fliC-specific primers. The kanamycin-resistance gene was then removed by transforming the strain with plasmid pCP20, which expresses the FLP recombinase, resulting in an in-frame deletion of the fliC gene. The fljB, spiB and ssaV genes were deleted in the same way but using the following primers: for fljB, 5'-TAACGTAACAGAGACAGCACGTTCTGCGGGACCTGGTTAGCCTGCGTGTAGGCTGGAGCTGCTTC-3' and 5'-TTATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCCATATGAATATCCTCCTTAGT-3'; for spiB, 5'-TTACAAGGCCGGGAAGTATGGCTGAATGAAGGTAACCTGTCACTGGTGTAGGCTGGAGCTGCTTC-3' and 5'-GGATAGTTAATCAAAGTATCATAATGTTTAATCGTTACCACATCGCATATGAATATCCTCCTTAGT-3'; and for ssaV, 5'-AAATTTCTGGAGTCGCAATGCGTTCATGGTTAGGTGAGGGAGTCAGTGTAGGCTGGAGCTGCTTC-3' and 5'-CATTGTCCGCCAACTCCTCTTCGCTAAGGTCAATACTTTCTACCACATATGAATATCCTCCTTAGT-3'. Plasmid pEG9127 is a derivative of pBAC108L and contains the cloned spiC gene (Uchiya et al., 1999). Bacteria were grown at 37 °C in Luria broth (LB). Kanamycin was used at 50 µg ml–1.
RNA preparation and quantitative real-time RT-PCR.
Bacteria were grown in LB. When the OD600 of the culture reached 1.8, total RNA was isolated using an RNeasy kit (Qiagen) in accordance with the manufacturer's protocol. The isolated RNA was treated with DNase I (Invitrogen) to remove contaminating DNA, and 2 µg RNA was reverse-transcribed with SuperScript II reverse transcriptase using random primers. Real-time PCRs were performed in 50 µl reaction mixtures containing 1 µl cDNA, 0.9 µM of each primer, 0.25 µM of each fluorescent probe, and TaqMan Universal Master Mix (Applied Biosystems). Amplification was carried out in 96-well optical plates on a 7300 Real-Time PCR system (Applied Biosystems) with an initial incubation for 2 min at 50 °C, followed by 10 min at 95 °C and then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The housekeeping gene 16S rRNA was used as an internal standard for quantification of the total RNA. The primer pairs and fluorescent probes were designed using Primer Express software version 3.0, and synthesized by Applied Biosystems. The specific fluorescent probes were labelled at the 5' end with the reporter dye 6-carboxyfluorescein (FAM). The sequences of the primer–probe combinations were as follows: for fliC, 5'-GGATACGCTGAATGTGCAACA-3', 5'-GAGTCACCTCACCGTTCGTCTTA-3', and 6-FAM-CAGGATATGCCGATACTA (fluorescent probe); and for 16S rRNA, 5'-AGATGGGATTAGCTTGTTGGTGA-3', 5'-GTAACGTCAATGCTGCGGTTA-3', and 6-FAM-CCACAACACCTTCCTC (fluorescent probe). Threshold cycle values were calculated from the amplification plots, and the amount of fliC gene expression was determined relative to the level of gene expression in wild-type Salmonella after both values were normalized to 16S rRNA levels. Each sample was analysed in triplicate.
Construction of fliC–lacZ fusion on a plasmid.
To construct a transcriptional fusion of the fliC promoter region to the promoterless lacZ gene in the promoter-probe vector pRL124 (Malo & Loughlin, 1988), a 0.45 kb DNA fragment containing the fliC promoter region was amplified by PCR using two primers, 5'-CGGGGTACCGGCTATTTCGCCGCCTAAGA-3' and 5'-CCGGAATTCGCTGTTAGCAGACTGAACCG-3'. The PCR product, digested with KpnI and EcoR1, was ligated into the corresponding sites of pRL124, producing pRL-fliC.
β-Galactosidase assay.
Bacteria were grown overnight in LB at 37 °C, and diluted 1 : 100 into fresh LB and grown with aeration to OD600 1.8. β-Galactosidase activity was measured with the substrate o-nitrophenyl β-D-galactoside, as described elsewhere (Miller, 1972). Each sample was assayed in triplicate.
Preparation of secreted and whole-cell proteins.
An overnight culture in LB was inoculated into 15 ml fresh LB at a 1 : 100 dilution. The cultures were grown at 37 °C with mild aeration to OD600 1.8. A 1 ml sample of the culture was centrifuged at 18 500 g for 15 min to remove bacterial cells or supramolecules such as flagella or pili. After filtration through a 0.2 µm pore-size filter (Advantec), filtrates were mixed with TCA (final concentration 6 %), placed on ice for 30 min, and centrifuged at 14 000 g for 20 min. After drying, the pellets were dissolved in 20 µl SDS sample buffer (50 mM Tris-HC1, pH 6.8, 2.5 % SDS, 10 % glycerol, 0.01 % bromophenol blue, 25 mM DTT) and boiled for 5 min. For preparation of whole-cell proteins, the bacterial pellet from the centrifugation step was suspended in 1 ml cold water and processed as described above. The pellets were dissolved in 200 µl SDS sample buffer and boiled for 5 min.
2D gel electrophoresis.
Secreted proteins from bacteria grown to OD600 1.8 were extracted and solubilized in rehydration buffer [8 M urea, 2 % 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 0.4 % immobilized pH gradient buffer (pH 3–10), 0.001 % bromophenol blue and 50 mM DTT]. After quantification of protein by the Bradford assay (Bio-Rad) with BSA as a standard, a total of 125 µl lysate containing 50 µg protein was applied to 7 cm IPG ReadyStrips covering a pH range from 3 to 6 (Bio-Rad). IEF of proteins was carried out with the PROTEAN IEF cell (Bio-Rad) using a preset method that allowed a minimum of 10 000 V-h. Focused strips were subjected to SDS-PAGE using a 10 % acrylamide gel and then stained with Bio-Safe Coomassie (Bio-Rad).
Protein identification by MALDI-TOF MS.
Protein spots were excised from the Coomassie-stained 2D gels, washed in 1 : 1 (v/v) 25 mM NH4HCO3/acetonitrile, and subjected to in-gel digestion with 50 ng trypsin µl–1 (Promega) overnight at 37 °C. The extracted peptides were desalted with a ZipTip column (Millipore) and then analysed by MALDI-TOF MS using a Voyager PK2 mass spectrometer (Applied Biosystems). Proteins were identified using the MASCOT program.
Cell culture and bacterial infection.
J774 E, a mannose-receptor-positive murine macrophage cell line, was maintained at 37 °C in an incubator containing 5 % CO2 in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10 % heat-inactivated fetal calf serum (HyClone), 100 U penicillin ml–1, and 100 µg streptomycin ml–1. The day before infection, the macrophages were plated at a density of 1.0x106 per well in six-well tissue-culture plates (Falcon) or 0.4x106 per well in 24-well plates in medium without antibiotics. Bacteria (25 per macrophage) were added, and the plates were centrifuged at 500 g for 10 min at room temperature. The cells were then incubated for 20 min at 37 °C to permit phagocytosis, and free bacteria were removed by three washes with PBS (Sigma-Aldrich). After washing, Dulbecco's modified Eagle's medium containing 12 µg gentamicin ml–1 was added to kill extracellular bacteria, after which the cells were incubated at 37 °C for the indicated times.
pcDNA3.1-fliC plasmid construction and transfection.
The fliC gene was amplified by PCR using the primers 5'-CGGGATCCATGGCACAAGTCATTAATACA-3' and 5'-CCGGAATTTTAACGCAGTAAAGAGAGGAC-3'. The PCR product was digested with BamHI and EcoRI and then ligated into the corresponding sites of pcDNA3.1(+) (Invitrogen), producing pcDNA3.1-fliC. The resulting construct was verified by direct sequencing. For transfection experiments, macrophages were plated at a density of 1.0x106 per well in six-well plates and then transfected with 2 µg pcDNA3.1-fliC plasmid using FuGENE HD transfection reagent (Roche) according to the manufacturer's protocol. After 30 h, cytosolic extracts were prepared from the macrophages and analysed by Western blotting. For controls, macrophages were transfected with the same amount of empty vector.
Immunofluorescence microscopy.
Macrophages were grown on glass coverslips in 24-well cell-culture plates and infected as described above. At 3 h after infection, samples were fixed in 3.7 % paraformaldehyde for 10 min and washed three times in PBS. Samples were then permeabilized in 0.2 % Triton X-100 for 10 min to allow entry of the primary antibodies, including rabbit polyclonal anti-Salmonella antibodies (Biodesign) and murine monoclonal anti-FliC antibodies (BioLegend). The secondary antibodies were Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 594-conjugated goat anti-mouse IgG. Samples were mounted onto glass slides using Vectashield (Vector laboratories) and viewed at x63 magnification on a confocal laser scanning microscope (LSM510 META, Zeiss).
Western blot analysis.
Western blot analyses were performed essentially as described previously (Uchiya & Nikai, 2004). The peptide fragment of the FliC and FljB polypeptides with a common sequence VQSANSTNSQSDLDSIQ was synthesized, and antiserum specific for the oligopeptide was obtained by immunization of rabbits with the peptide coupled to keyhole limpet haemocyanin using benzidine. The resulting anti-flagellin peptide antibody was used at a dilution of 1 : 300. Cell lysates from macrophages infected with the respective Salmonella strains were prepared, and detection of SOCS-3 protein and the three MAPKs, including extracellular signal-regulated kinase (ERK), p38 and c-Jun amino-terminal kinase (JNK), was performed as described previously (Uchiya & Nikai, 2005). IB-
was detected with a 1 : 500 dilution of anti-IB- antibody (Santa Cruz Biotechnology).
Statistical analysis.
Each experiment was performed at least three times. The results are expressed as means±SDs. The data were analysed by analysis of variance with Dunnett's test. A value of P<0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, indicated by black arrowheads) that was more intense in the secreted proteins from the wild-type strain than in those from the spiC mutant. The corresponding spots were excised from the gel, digested with trypsin, and analysed by MALDI-TOF MS. The MASCOT program was employed for the identification of the protein spots. Search results using the NCBlnr protein database showed that protein scores greater than 60 were significant (P<0.05), and there was only one protein with a protein score of 70. MASCOT identified both spots as containing the flagellin protein FliC, a component of flagellar filaments, suggesting that the absence of SpiC results in a reduction in the level of secreted FliC.
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). Because these flagellin proteins are exported into the medium from the cytoplasm via the flagellum-specific type III export system (Komoriya et al., 1999; Macnab, 1996), we suspected that SpiC participates in either the transport or the expression of flagellin proteins. To examine these possibilities, we grew the bacteria to OD600 1.8 in LB to induce the expression of the spiC gene, and then analysed the proteins both in the medium and contained in the cells by Western blotting with a rabbit anti-flagellin antiserum that recognizes a peptide common to FliC and FljB. From their amino acid sequences, FliC and FljB are predicted to be 51.6 and 52.5 kDa, respectively. We observed bands corresponding to FliC and FljB in the wild-type strain. The identities of the two bands were also confirmed by comparison of the patterns from the wild-type, fliC, fljB and double-mutant strains (Fig. 2). Although a similar amount of FljB was detected in the wild-type strain and the spiC mutant, in both the culture supernatant and whole-cell lysates, the level of FliC was much lower in the spiC mutant than in the wild-type strain. Complementation of the spiC mutant with a plasmid carrying the wild-type spiC gene (pEG9127) restored FliC to the level found in the wild-type strain. These results demonstrated that SpiC participates in the expression but not the transport of FliC protein.
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) and by quantitative real-time RT-PCR (Fig. 3b). In both assays, the expression level of the fliC gene in the spiC mutant was greatly reduced compared with the wild-type strain, indicating that SpiC participated in the regulation of FliC transcription.
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), which is a two-component regulatory gene involved in the expression of SPI-2-encoded genes, resulted in defective expression of the fliC gene, similar to the spiC mutant (Fig. 3c
); however, the defective phenotype was not seen in the spiB and ssaV mutants, which lack a putative component of the SPI-2 TTSS (Ochman et al., 1996; Hensel et al., 1997). These results suggested the specific involvement of SpiC in the expression of the fliC gene.
Involvement of FliC in the induction of SOCS-3 expression in Salmonella-infected macrophages
SOCS-3 is known to inhibit cytokine signalling via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway (Kile & Alexander, 2001; Suzuki et al., 2001). Our previous studies showed that SpiC participates in the induction of SOCS-3 expression by affecting the signal transduction pathways in Salmonella-infected macrophages (Uchiya & Nikai, 2005); however, the mechanism by which SpiC mediates activation of the signal transduction pathways in macrophages after Salmonella infection remains unknown. Because we found that SpiC contributes to the expression of FliC, we first examined the involvement of FliC in the induction of SOCS-3 expression in macrophages using two methods. In one set of experiments, flagellin (FliC) isolated from S. enterica serovar Typhimurium strain 14028 (Alexis) was added to cell cultures, and in the other set of experiments, macrophages were transfected with the pcDNA3.1-fliC plasmid, which carries the wild-type Salmonella fliC gene. After treatment, cell lysates were prepared and the expression of SOCS-3 was assessed by Western blotting. Treatment of macrophages with purified FliC induced the dose-dependent expression of SOCS-3 when measured 1.5 h post-treatment (Fig. 4a). The extent of induction was lower 3 h post-treatment. Furthermore, transfection of macrophages with plasmid pcDNA3.1-fliC resulted in an approximately 1.8-fold increase in SOCS-3 expression compared with the vector control (Fig. 4b), indicating that FliC can induce SOCS-3 expression.
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shows that, at 2.5 h post-infection, the levels of SOCS-3 in fliC mutant-infected macrophages were reduced by up to 50 % compared with the levels in wild-type Salmonella-infected macrophages, and that the level of SOCS-3 induced by the fliC/fljB double mutant was twofold lower than that induced by the fliC mutant. Similar patterns of SOCS-3 expression were also observed at 8 h post-infection, although there were no significant differences at 1 h post-infection. A mutation of the fljB gene, however, had little effect on the expression of SOCS-3. Taken together with the results from Figs 2 and 3, these findings demonstrate the involvement of FliC in SPI-2-dependent induction of SOCS-3 expression. Furthermore, the expression of SOCS-3 in macrophages infected with the fliC mutant did not decrease to the level seen for the spiC mutant-infected macrophages. This result indicates that FliC contributes to SPI-2-dependent induction of SOCS-3 expression but that there must also be FliC-independent mechanisms.
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). Therefore, we examined the involvement of FliC in the activation of MAPK pathways by measuring the levels of phosphorylated MAPK in Salmonella-infected macrophages. Western blotting using phospho-specific antibodies (Fig. 6) revealed that the levels of MAPK phosphorylation in Salmonella-infected macrophages corresponded with the levels of SOCS-3 expression. Infection of macrophages with the wild-type strain resulted in higher levels of phosphorylated ERK, p38 and JNK than did infection with the spiC mutant, indicating that SpiC participates in the activation of the MAPK pathways in Salmonella-infected macrophages. The levels of MAPK phosphorylation in the fliC-mutant-infected macrophages were lower than in the wild-type strain-infected macrophages, but the levels were not as low as those in the spiC mutant-infected macrophages, indicating that the fliC mutant is weaker in the activation of the MAPK pathways than the spiC mutant. Taken together, these results indicate that FliC partially participates in the SPI-2-dependent expression of SOCS-3 through the activation of the MAPK pathways.
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). In addition, the defective expression in the spiC mutant could be rescued by introducing pEG9127, which carries the spiC gene. These results suggest that the presence of FliC-derivative filaments inside Salmonella-infected macrophages is important for the induction of SOCS-3 expression.
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B pathway participates in SpiC-dependent expression of SOCS-3B pathway in epithelial cells (Ogushi et al., 2001; Reed et al., 2002; Takahashi et al., 2001) and because the NF-B pathway participates in the induction of SOCS-3 expression (Hayashi et al., 2002), we investigated the participation of the NF-B signalling pathway in SPI-2-dependent expression of SOCS-3. As shown in Fig. 8(a), MG-132, a NF-B inhibitor, dose-dependently reduced the expression of SOCS-3 in wild-type Salmonella-infected macrophages, suggesting the importance of the NF-B pathway in Salmonella-induced SOCS-3 expression. To further confirm the participation of the NF-B pathway, we measured the level of NF-B activation in Salmonella-infected macrophages. In resting cells, NF-B makes a complex with the inhibitory protein IB in the cytoplasm (Baldwin, 1996). Upon appropriate cell stimulation, IB is rapidly phosphorylated, ubiquitinated and then degraded by the 26S proteasome (Brown et al., 1995; May & Ghosh, 1998). This degradation of IB frees NF-B to enter the nucleus and stimulate the transcription of target genes. Therefore, we followed the activation of NF-B by measuring the level of IB- degradation. The experiments were performed in the presence of cycloheximide (0.1 µg ml–1) to block the synthesis of new IB-. LPS, an activator of NF-B, was used as a positive control. As shown in Fig. 8(b), the levels of IB- protein in the wild-type strain-infected macrophages at 2.5 and 5 h post-infection were decreased up to 60 and 45 % compared with those of uninfected macrophages, respectively, whereas infection with the spiC mutant had little effect on the level of IB- protein. Taken together, these results demonstrate that the NF-B pathway plays a significant role in SPI-2-dependent expression of SOCS-3. In contrast, the fliC mutant and fliC/fljB double mutant caused a similar amount of IB- degradation to that of the wild-type strain, indicating that FliC does not participate in the activation of NF-B in Salmonella-infected macrophages.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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SOCS-3 is a member of the SOCS family, which includes SOCS-1 to -7 and Shc (Starr et al., 1997; Yoshimura et al., 1995). These SOCS proteins act as negative regulators of the JAK/STAT signalling pathway. Ligand binding by cytokine receptors activates JAK, which then phosphorylates STAT proteins. This phosphorylation by JAK is required for the dimerization of STAT and its nuclear translocation, binding of DNA, and activation of gene transcription (Kile & Alexander, 2001; Yasukawa et al., 1999). In addition to inhibiting the response to IL-6 by blocking the phosphorylation of STAT-3, SOCS-3, like SOCS-1, has been reported to inhibit IFN- signalling by blocking STAT-1 phosphorylation (Song & Shuai, 1998; Stoiber et al., 1999). Cytokines, especially IFN-, are essential contributors to macrophage activation, promoting the effective killing of certain intracellular pathogens (Bhardwaj et al., 1986; Green et al., 1990; Suzuki et al., 1988; van Dissel et al., 1987). This suggests that the expression of SOCS-3 affects host defences against intracellular pathogens by inhibiting IFN- signalling. In our previous studies, cDNA array analysis revealed that the expression of SOCS-3 in Salmonella-infected macrophages is upregulated in a SPI-2-dependent manner, leading to the inhibition of IL-6 and IFN- signalling via the JAK/STAT signalling pathway (Uchiya & Nikai, 2005). This suggested that the expression of SOCS-3 is important for the virulence of Salmonella; however, the mechanism by which SpiC mediates the induction of SOCS-3 expression in macrophages after Salmonella infection remains unknown. It has been reported that SOCS-3 expression is stimulated not only by cytokines but also by PAMPs, such as LPS, bacterial CpG-DNA, and Gram-positive bacteria (Bode et al., 1999; Dalpke et al., 2001; Stoiber et al., 1999, 2001). In the present study, we focused on the role of flagella, which are abundant on the bacterial surface, in SPI-2-dependent expression of SOCS-3.
The flagellum is composed of a basal body, a hook and a filament. Flagellin protein, a component of the filament, is transported from the cytoplasm by the flagellum-specific type III export system (Komoriya et al., 1999) and is polymerized with the help of the cap protein FliD, producing long helical flagellar filaments (Ikeda et al., 1996). S. enterica serovar Typhimurium expresses two antigenically distinct flagellins encoded by the fliC and fljB genes. The alternative expression of these two genes is known as phase variation, and it occurs on a timescale of the order of 103–105 generations (Silverman, 1979). In the present study, proteomic analysis revealed that the level of FliC protein is much lower in the culture supernatant from the spiC mutant than in that from the wild-type strain. We further investigated the mechanism by which SpiC alters the levels of secreted FliC. SpiC is required for secretion of some virulence factors from the cytoplasm by the SPI-2 TTSS (Freeman et al., 2002; Yu et al., 2002), although the molecular mechanism is not known. Several genes that encode the SPI-2 TTSS and the flagellum-specific type III export system have sequences in common (Aizawa, 2001; Macnab, 2004). These findings suggest that, in addition to its role in SPI-2 TTSS, SpiC might participate in the export of flagellin proteins from the cytoplasm via the type III flagellar protein export system. Our results showed that the levels of FliC were reduced not only in the culture supernatant but also in whole-cell lysates of the spiC mutant, indicating that the reduced level of FliC was due to altered expression rather than an inability to transport FliC via the type III flagellar protein export system. Furthermore, quantitative real-time RT-PCR revealed that this mutant had a much lower level of fliC mRNA. These results indicate that SpiC controls the expression of FliC at the transcriptional level. A gel-shift assay did not provide evidence that SpiC binds to the promoter region of the fliC gene (data not shown), which suggests that SpiC participates indirectly in the induction of fliC mRNA expression. We further examined the possibility that SpiC alters flagellar phase variation by using the fljB/fljA mutant in which phase variation is eliminated. The fljB gene constitutes an operon with the fliA gene, which encodes a negative regulator of fliC expression (Yamamoto & Kutsukake, 2006). The expression level of FliC in the fljB/fljA mutant was decreased by a mutation of the spiC gene (data not shown). This suggests that SpiC is not involved in phase variation, although we cannot completely rule out this possibility. Taken together, these results suggest that SpiC affects the expression of the class 1 gene flhDC or the class 2 gene fliA (Macnab, 1996), which, respectively, function as a master regulator of flagellar gene expression and a regulator of class 3 gene expression. Further studies are needed to clarify how SpiC contributes to the expression of the fliC gene.
The flagellar filament protein FliC is a potent stimulator of immune and proinflammatory responses (Gewirtz et al., 2001b; Hayashi et al., 2001). There have been many reports that it activates signal transduction pathways via TLR5 in cultured cells such as epithelial cells, leading to the induction of immune and proinflammatory genes (Eaves-Pyles et al., 2001; Gewirtz et al., 2001a; Sierro et al., 2001; Zeng et al., 2003). Okugawa et al. (2006) reported the induction of SOCS-1 by flagellin stimulation in Jurkat T cells. As described above, SpiC contributes to the expression of FliC, suggesting that FliC participates in SpiC-dependent expression of SOCS-3. As expected, we found that the level of SOCS-3 expression in macrophages infected with the fliC mutant was lower than that of the wild-type strain-infected macrophages, whereas a mutation of the fljB gene had little effect on SOCS-3 expression. These results indicate that FliC plays a significant role in the SpiC-dependent expression of SOCS-3. The level of SOCS-3 expression in the fliC mutant-infected macrophages, however, did not drop to that in spiC mutant-infected macrophages, suggesting the existence of additional FliC-independent mechanisms in the SPI-2-dependent expression of SOCS-3.
In subsequent studies, we focused on the role of FliC in the signal transduction pathways governing the SPI-2-induced expression of SOCS-3. SPI-2-dependent SOCS-3 expression is reported to occur downstream of the ERK and p38 MAPK signalling pathways (Uchiya & Nikai, 2005). In the present study, we showed that, in addition to ERK and p38 MAPK, the NF-B pathway contributes to the SPI-2-dependent activation of SOCS-3 expression. In contrast, although FliC was involved in the activation of MAPK pathways, we could not find evidence for the involvement of FliC in NF-B activation, indicating that FliC is not required for all aspects of SPI-2-dependent activation of the signal transduction pathways. Flagellin is recognized by TLR5, and ligand binding by TLR5 leads to NF-B activation (Hayashi et al., 2001; Zeng et al., 2003). Therefore, our results suggest that FliC activates TLR5-independent signal transduction pathways in macrophages. In addition to TLR5, flagellin was recently shown to be recognized in the cytosol by two different nucleotide-binding oligomerization domain (Nod)-like receptors, Ipaf and Naip5 (also known as Birc1e) (Molofsky et al., 2006; Ren et al., 2006). Miao et al. (2006) and Franchi et al. (2006) have reported that flagellin activates a signalling pathway independent of TRL5, which leads to caspase-1 cleavage and the secretion of IL-1β via Ipaf in the cytosol of Salmonella-infected macrophages. These findings indicate that Ipaf plays an important role in the induction of SOCS-3 expression by FliC in Salmonella-infected macrophages. This possibility is supported by our finding that SOCS-3 expression is elevated in pcDNA3.1-fliC-transfected macrophages.
There are several reports that the expression of FliC is downregulated in Salmonella-infected macrophages or during Salmonella infection in mice (Alaniz et al., 2006; Cummings et al., 2006; Eriksson et al., 2003). However, our data showed that FliC was detected in about 70 % of macrophages infected with the wild-type strain but almost not at all in macrophages infected with the spiC mutant. Lyons et al. (2004) reported that infection of polarized epithelial cells by Salmonella leads to IL-8 expression by causing the SPI-2-dependent translocation of flagellin to a basolateral membrane domain expressing TLR5. Together with our results, this suggests that SPI-2 mediates not only the transcytosis of flagellin in infected cells but also its expression.
In conclusion, we showed that SpiC is required for the transcriptional expression of flagellin in S. enterica serovar Typhimurium. We also demonstrated that the SpiC-dependent expression of FliC activates MAPK signalling pathways in macrophages after Salmonella infection, leading to the induction of SOCS-3 expression. It is likely that these events play important roles in the pathogenesis of Salmonella infection.
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ACKNOWLEDGEMENTS |
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Edited by: J. G. Shaw
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REFERENCES |
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Received 24 June 2008;
revised 6 August 2008;
accepted 7 August 2008.
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