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Research Article
21 February 2019

Salmonella Fimbrial Protein FimH Is Involved in Expression of Proinflammatory Cytokines in a Toll-Like Receptor 4-Dependent Manner

ABSTRACT

Type 1 fimbriae are proteinaceous filamentous structures present on bacterial surfaces and are mainly composed of the major fimbrial protein subunit FimA and the adhesive protein FimH, which is located at the tip of the fimbrial shaft. Here, we investigated the involvement of type 1 fimbriae in the expression of proinflammatory cytokines in macrophages infected with Salmonella enterica serovar Typhimurium. The level of interleukin-1β (IL-1β) mRNA was lower in macrophages infected with fimA or fimH mutant strains than in those infected with wild-type Salmonella. Treatment of macrophages with purified recombinant FimH protein, but not FimA, resulted in the activation of the mitogen-activated protein kinase and nuclear factor κB signaling pathways, leading to the expression of not only IL-1β but also other proinflammatory cytokines, such as IL-6 and tumor necrosis factor alpha. However, FimH carrying an N-terminal region deletion or heat-treated FimH did not show such effects. The expression of FimH-induced IL-1β was inhibited by treatment with the Toll-like receptor 4 (TLR4) inhibitor TAK-242 but not by treatment with polymyxin B, a lipopolysaccharide antagonist. Furthermore, FimH treatment stimulated HEK293 cells expressing TLR4 and MD-2/CD14 but did not stimulate HEK293 cells expressing only TLR4. Collectively, FimH is a pathogen-associated molecular pattern of S. enterica serovar Typhimurium that is recognized by TLR4 in the presence of MD-2 and CD14 and plays a significant role in the expression of proinflammatory cytokines in Salmonella-infected macrophages.

INTRODUCTION

Salmonellae are widespread Gram-negative bacteria with fimbriae and peritrichous flagella that cause a variety of disease syndromes in humans and animals (1). Salmonella enterica serovar Typhimurium causes gastroenteritis in humans and a systemic disease in mice similar to human typhoid fever. Salmonellae are transmissible via the fecal-oral route. After reaching the intestinal lumen, salmonellae invade and destroy specialized epithelial cells in the host’s intestine and migrate to the mesenteric lymph nodes, where they encounter macrophages that play an important role in host defense (2). However, despite the various bactericidal mechanisms deployed in macrophages, salmonellae can survive and replicate within macrophages. Specific virulence factors encoded within Salmonella pathogenicity islands (SPIs) are required at various stages of Salmonella infection (3). Among these virulence factors, SPI-1 and SPI-2 play crucial roles in the invasion of host cells and intracellular survival, respectively (47). SPI-1 is also implicated in the onset of inflammatory diarrhea via a mechanism involving modulation of inflammatory responses in enteric tissue (810). This event is considered important in intestinal colonization by Salmonella (11, 12).
Type 1 fimbriae are proteinaceous filamentous structures that are present on the surface of many members of the Enterobacteriaceae, including S. enterica and Escherichia coli, and that mediate mannose-sensitive bacterial adhesion to target cells (13, 14). Type 1 fimbriae are mainly composed of the protein subunits FimA and FimH. The adhesive properties of type 1 fimbriae depend on adhesion by lectin-like FimH, which is located at the tip of the fimbrial shaft. FimH is responsible for binding to target receptors and exhibits specificity for glycoproteins containing terminal mannose residues (1517). This adhesive protein has been reported to play an important role in bacterial adhesion during the process of colonization and invasion of host tissue (1823). Moreover, the interactions between type 1 fimbriae and host cell receptors play a critical role in the initiation and modulation of innate and adaptive immune responses (2426).
Pathogen-associated molecular patterns (PAMPs), which are components of bacteria and viruses, induce the expression of mediators by several types of cells, including macrophages, and influence the host immune system (27). PAMPs include cell wall components derived from Gram-positive bacteria, lipopolysaccharides (LPS) from Gram-negative bacteria, lipoteichoic acid, flagella, and fimbriae, all of which are recognized by pattern recognition receptors such as Toll-like receptors (TLRs). TLRs comprise a large family of proteins with characteristic extracellular leucine-rich repeats and a cytoplasmic Toll–interleukin-1 (IL-1) receptor homology domain and have been implicated in the recognition of PAMPs (28). Ligand binding by TLRs initiates intracellular signaling and induces the expression of immune and inflammatory genes. These signaling pathways can be divided into MyD88-dependent and MyD88-independent cascades (29). Specifically, bacterial cell wall components and lipoteichoic acid are recognized by TLR2 (30), and LPS and the flagellar filament protein FliC then bind TLR4 and TLR5, respectively (3133). Furthermore, there are reports that certain bacterial fimbriae activate signal transduction pathways via particular TLRs in cultured cells and lead to the induction of proinflammatory genes (26, 34, 35).
In this study, we investigated the involvement of type 1 fimbriae in the expression of proinflammatory cytokines in macrophages infected with S. enterica serovar Typhimurium. We found that Salmonella FimH, but not FimA, is a PAMP that is recognized by TLR4 and plays a significant role in the expression of proinflammatory cytokines in Salmonella-infected macrophages.

RESULTS

Involvement of FimH in the expression of IL1B mRNA in macrophages.

Type 1 fimbriae are proteinaceous filamentous structures that are composed mainly of FimA and FimH proteins. We constructed two mutant strains carrying deletions of fimA and fimH to examine the participation of type 1 fimbriae in the expression of proinflammatory cytokines in macrophages infected with S. enterica serovar Typhimurium. To confirm the formation of type 1 fimbriae on the fimA and fimH mutants, hemagglutination assays were performed in the presence and absence of d-mannose. The wild-type strain showed obvious hemagglutination in the absence of mannose that was then inhibited in the presence of mannose, whereas the fimA and fimH mutants displayed no visible hemagglutination activity (data not shown). We examined whether these fimbrial genes participate in IL-1β (IL1B) expression in the mouse macrophage cell line J744 infected with S. enterica serovar Typhimurium. IL1B expression was analyzed by quantitative real-time PCR (qRT-PCR) of total RNA extracted from macrophages at 5 h postinfection. To reduce disparities in bacterial adhesion, we promoted adhesion of the bacteria to the macrophages by centrifugation, as stated in Materials and Methods. Indeed, almost no difference was observed between the uptake of bacteria by macrophages infected with the wild type and that of macrophages infected with mutant Salmonella strains (see Fig. S1 in the supplemental material). As shown in Fig. 1A, the level of IL1B mRNA in macrophages was lower when they were infected with the fimA or fimH mutant than when they were infected with wild-type Salmonella. These results indicated that type 1 fimbriae could be involved in IL1B expression in Salmonella-infected macrophages.
FIG 1
FIG 1 FimH, but not FimA, induces IL1B mRNA expression in macrophages. (A) Involvement of type 1 fimbriae in induction of IL1B expression in Salmonella-infected macrophages. Macrophages were infected with wild-type (WT), fimA mutant, or fimH mutant Salmonella. At 5.0 h postinfection, total RNA was prepared and analyzed by qRT-PCR. Abundances of IL1B mRNA were normalized to those of GAPDH mRNA. *, P < 0.001 (significantly different from macrophages infected with wild-type Salmonella). UI, uninfected. (B) SDS-PAGE of purified recombinant proteins stained with Coomassie brilliant blue. Lane 1, FimA; lane 2, FimH; lane 3, FimH-C (C-terminal domain of FimH). Molecular masses of standard proteins are indicated on the left. (C) Involvement of FimH in induction of IL1B expression in macrophages. Macrophages were treated with purified recombinant proteins at the indicated concentrations. After 2.5 h of treatment, total RNA was prepared and analyzed by qRT-PCR. *, P < 0.001 (significantly different from untreated [UT] macrophages).
Next, to examine the direct contribution of type 1 fimbrial protein to IL1B expression, FimA and FimH proteins were purified as described in Materials and Methods. The purified recombinant proteins appeared as single bands of approximately 24.8 kDa for FimA and 38.5 kDa for FimH on gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1B). We then examined the involvement of FimA and FimH in the induction of IL1B expression in macrophages. After macrophages were treated for 2.5 h with FimA (10 μg/ml) or FimH (2.5 and 5.0 μg/ml), total RNA was prepared, and the expression of IL1B mRNA was assessed by qRT-PCR. Treatment of macrophages with FimH induced dose-dependent expression of IL1B (Fig. 1C). On the other hand, FimA did not induce IL1B expression, even when applied at double the concentration of FimH, indicating the involvement of FimH, but not FimA, in the induction of IL1B mRNA expression in macrophages.
To investigate the structure-activity properties of FimH, we purified the 152-amino-acid recombinant FimH protein (amino acid positions 161 to 312) without its N-terminal region (Fig. 1B). This truncated recombinant protein did not induce IL1B expression (Fig. 1C), suggesting that the N terminus of FimH is necessary for the expression of its activity, but its C terminus is not.
We further examined whether other proinflammatory cytokines, such as IL-6 (IL6) and tumor necrosis factor alpha (TNF-α) (TNFA), were induced in FimH-treated macrophages. After treatment, the expression of both IL6 and TNFA was assessed by qRT-PCR. As shown in Fig. 2, the expression levels of the mRNAs encoding both cytokines increased, indicating that FimH can induce the expression of not only IL1B but also IL6 and TNFA.
FIG 2
FIG 2 Expression of IL6 and TNFA mRNAs in macrophages treated with FimH or FimA. Macrophages were treated with FimH (2.5 or 5.0 μg/ml) or FimA (10 μg/ml). After 2.5 h of treatment, total RNA was prepared and analyzed by qRT-PCR. (A) Quantitative analysis of IL6 mRNA expression. (B) Quantitative analysis of TNFA mRNA expression. Abundances of IL6 and TNFA mRNAs were normalized to those of GAPDH mRNA. *, P < 0.01; **, P < 0.001 (significantly different from untreated [UT] macrophages).

FimH participates in IL1B mRNA expression via activation of NF-κB and p38 MAPK signaling pathways in macrophages.

Subsequent experiments focused on the signal transduction pathways that govern FimH-induced expression of IL1B. We first examined the involvement of the nuclear factor κB (NF-κB) signal transduction pathway in the expression of IL1B. As shown in Fig. 3A, although no major decrease in IL1B expression was observed upon the addition of the NF-κB inhibitor MG-132, MG-132 significantly reduced FimH-induced expression of IL1B. This result indicates that the NF-κB pathway could participate in FimH-induced expression of IL1B. To confirm whether the NF-κB pathway participates in IL1B expression, we estimated NF-κB activation by measuring the level of IκB-α degradation in FimH-treated macrophages. In resting cells, NF-κB complexes with the inhibitory protein IκB in the cytoplasm (36). Upon appropriate cell stimulation, IκB is rapidly phosphorylated, ubiquitinated, and then degraded by the 26S proteasome (37, 38). This degradation of IκB frees NF-κB to enter the nucleus and stimulate the transcription of target genes. To monitor NF-κB activation by measuring IκB-α degradation, experiments were performed in the presence of cycloheximide (0.1 μg/ml) to block the synthesis of new IκB-α. LPS, an activator of NF-κB, was used as a positive control. As shown in Fig. 3B, IκB-α protein quantities in FimH-treated macrophages at 2.5 h posttreatment decreased in a dose-dependent manner compared with those in untreated macrophages. These findings demonstrate that the NF-κB pathway is involved in FimH-induced IL1B expression.
FIG 3
FIG 3 Involvement of the NF-κB signaling pathway in FimH-induced IL1B mRNA expression in macrophages. (A) Effect of the NF-κB inhibitor MG-132 on FimH-induced expression of IL1B mRNA. Macrophages were treated with FimH (2.5 μg/ml) in the presence of the indicated concentrations of the inhibitor or 0.1% dimethyl sulfoxide alone (solvent control). After 2.5 h of treatment, total RNA was prepared and analyzed by qRT-PCR. Abundances of IL1B mRNA were normalized to those of GAPDH mRNA. *, P < 0.01; **, P < 0.001 (significantly different from macrophages not treated with the inhibitor). (B) Degradation of IκB-α in macrophages treated with FimH or LPS. Macrophages were treated with FimH (2.5 or 5.0 μg/ml) or LPS (1.0 μg/ml) as a positive control in the presence of cycloheximide (0.1 μg/ml). At 2.5 h postinfection, cytosolic extracts were prepared, and IκB-α degradation was analyzed using an anti-IκB-α antibody. IκB-α quantities were normalized to those of actin. Graphs show values as percentages compared with untreated (UT) macrophages. *, P < 0.01; **, P < 0.001 (significantly different from untreated macrophages).
We next examined the involvement of the mitogen-activated protein kinase (MAPK) signal transduction pathways on the expression of IL1B. Figure 4 shows that IL1B expression in FimH-treated macrophages was blocked by SB203580, which inhibits the p38 MAPK in a concentration-dependent manner, but not by the extracellular signal-regulated kinase (ERK) inhibitor PD98059 or the c-Jun amino-terminal kinase (JNK) inhibitor SP600125. To confirm the participation of the MAPK pathways, we estimated MAPK activation by quantifying phosphorylated p38, ERK, and JNK MAPK levels in FimH-treated macrophages. As shown in Fig. 5, treatment of macrophages with FimH increased p38, ERK, and JNK MAPK phosphorylation in a dose-dependent manner when measured at 2.5 h posttreatment. Furthermore, because FimH-induced IL1B expression was not affected by PD98059 or SP600125 (Fig. 4), we also confirmed that these inhibitors could effectively inhibit the ERK and JNK MAPK pathways. At the concentration used in our study, PD98059 and SP600125 showed approximately 80% and 70% inhibition of FimH-induced ERK and JNK MAPK phosphorylation, respectively (see Fig. S2 in the supplemental material). This result shows that both inhibitors could effectively inhibit their respective MAPK pathways. Taken together with the results from Fig. 3, these findings demonstrate that the NF-κB and p38 MAPK pathways play significant roles in FimH-induced expression of IL1B.
FIG 4
FIG 4 Effects of inhibitors of ERK (PD98059), p38 (SB203580), and JNK (SP600125) on FimH-induced IL1B expression in macrophages. Macrophages were treated with FimH (2.5 μg/ml) in the presence of the indicated concentrations of inhibitors or 0.1% dimethyl sulfoxide (solvent control). After 2.5 h of treatment, total RNA was prepared and analyzed by qRT-PCR. Levels of IL1B mRNA were normalized to those of GAPDH mRNA. *, P < 0.001 (significantly different from macrophages not treated with an inhibitor). UT, untreated.
FIG 5
FIG 5 Western blot analysis of phospho-ERK, phospho-p38, and phospho-JNK in macrophages treated with FimH. Cytosolic extracts were prepared 2.5 h after treatment of cells with FimH (2.5 μg/ml) and were analyzed with the indicated antibodies. (A) Image of the original blots. After analysis with anti-phospho-ERK, -phospho-p38, and -phospho-JNK (top), the membranes were stripped and reprobed with an antibody to ERK, p38, or JNK (bottom). (B) Densitometric analysis of the amounts of phospho-ERK, phospho-p38, and phospho-JNK normalized to the amounts of ERK (p44), p38, and JNK (p54) in the same samples. Graphs show values as percentages compared with untreated macrophages. *, P < 0.01; **, P < 0.001 (significantly different from untreated macrophages).

TLR4 is necessary for FimH-induced IL1B mRNA expression.

We next explored the mechanism by which FimH induces IL1B expression in macrophages. Many ligands bind with TLRs, which play a major role in pathogen recognition and the initiation of inflammatory and immune responses (27). We focused on TLR4 as a receptor for FimH and examined the involvement of TLR4 in FimH-induced IL1B expression. Treatment of macrophages with LPS, a TLR4 agonist, results in an increase in IL1B expression at 2.5 h posttreatment. This increase was inhibited by treatment with TAK-242, a TLR4 inhibitor (Fig. 6A), indicating that stimulation of TLR4 by LPS can induce IL1B expression in macrophages. Blocking of TLR4 with TAK-242 also showed that TLR4 was responsible and necessary for FimH-induced IL1B expression (Fig. 6A).
FIG 6
FIG 6 TLR4 is necessary for FimH-induced IL1B expression. (A) Inhibition of FimH-induced IL1B expression in macrophages by TAK-242. Macrophages were pretreated for 1 h with TAK-242 (40 μM) and then stimulated with FimH (2.5 μg/ml) or LPS (1.0 μg/ml) for 2.5 h. **, P < 0.001 (significantly different from macrophages not treated with TAK-242). (B) Effect of polymyxin B (PMB) on FimH-induced IL1B expression. Macrophages were stimulated for 2.5 h with FimH (2.5 μg/ml) or LPS (1.0 μg/ml) that had been pretreated with or without polymyxin B (100 μg/ml) for 1 h at 37°C. (C) Macrophages were stimulated for 2.5 h with FimH (2.5 μg/ml) or LPS (1.0 μg/ml) that had been treated at 100°C for 30 min. Total RNA was isolated from each type of cell for qRT-PCR analysis. *, P < 0.01; **, P < 0.001 (significantly different from macrophages treated with unheated FimH or LPS). (D) FimH is recognized by TLR4 in the presence of MD-2 and CD14. HEK293 cells, HEK293 cells expressing mouse TLR4 (HEK293/mTLR4), and HEK293 cells expressing mouse TLR4 and MD-2/CD14 (HEK293/mTLR4/MD-2/CD14) were transiently transfected with pNiFty-Luc and pRL-SV40 and then stimulated with FimH (2.5 μg/ml) or LPS (10 ng/ml). At 6 h posttreatment, cell lysates were harvested, and luciferase activity was measured. Firefly luciferase signals associated with NF-κB were normalized to internal Renilla luciferase levels as a transfection control. **, P < 0.001 (significantly different from untreated [UT] cells).
We also investigated the possibility that TLR4-mediated induction of IL1B expression by FimH could be an artifact of LPS contamination in the purified FimH protein solution in the following ways. First, we tested the level of LPS in the purified FimH solution using a Limulus amoebocyte lysate assay. This sample was contaminated with 48.8 pg of LPS per 1 μg of FimH protein, which is equivalent to 122 pg of LPS per 2.5 μg of FimH. Treatment of macrophages with FimH (2.5 μg/ml) induced 5- to 6-fold increased expression levels of IL1B compared with those in untreated cells (Fig. 1C), whereas treatment with LPS at 1.0 ng/ml did not induce increased IL1B expression (data not shown). Second, we investigated the effects of polymyxin B, a neutralizer of LPS (39), on the induction of IL1B expression in macrophages by FimH. As shown in Fig. 6B, polymyxin B at 100 μg/ml did not affect FimH-induced IL1B expression and completely blocked that induced by LPS. Third, we examined the effect of heat treatment on the ability of FimH to induce IL1B expression in macrophages. As shown in Fig. 6C, LPS treatment increased IL1B expression even after a 30-min heat treatment at 100°C, while the effect of FimH on IL1B expression was largely abolished by heat treatment. This result shows that the ability of FimH to induce IL1B expression is not thermostable, unlike that of LPS. Together, these results strongly suggest that the induction of TLR4-mediated IL1B expression by FimH is not an artifact due to LPS contamination.
To further confirm the participation of TLR4 in the induction of IL1B expression by FimH, we examined the ability of FimH to stimulate human embryonic kidney HEK293 cells, HEK293 cells expressing mouse TLR4 (HEK293/mTLR4), and HEK293 cells expressing mouse TLR4 and MD-2/CD14 (HEK293/mTLR4/MD-2/CD14). MD-2 and CD14 are known to associate with the extracellular domain of TLR4 and augment TLR4-dependent LPS responses (29, 40, 41). The above-mentioned cells were transfected with the pNiFty-Luc NF-κB firefly luciferase reporter construct and the pRL-SV40 Renilla luciferase reporter vector as a control for transfection efficiency. The transfected cells were then stimulated with FimH or LPS for 6 h, and luciferase activity was measured as described in Materials and Methods. As shown in Fig. 6D, the HEK293/mTLR4/MD-2/CD4 cells responded to FimH, as well as LPS, with a significant increase in luciferase activity compared with that observed in HEK293 cells. On the other hand, luciferase activity after LPS treatment in HEK293/mTLR4 cells was approximately one-fourth of that in HEK293/mTLR4/MD-2/CD4 cells, and luciferase activity after FimH treatment was comparable to that in untreated cells. Taken together with the results shown in Fig. 6A, these results confirmed that FimH stimulates luciferase activity through TLR4 and that MD-2 and CD14 are essential components of the TLR4-mediated stimulation of luciferase activity by FimH.

DISCUSSION

In this study, we show that type 1 fimbriae are involved in the expression of proinflammatory cytokines in macrophages infected with S. enterica serovar Typhimurium. We also demonstrate that purified recombinant FimH protein, but not FimA, results in the activation of the MAPK and NF-κB signaling pathways via TLR4, leading to the expression of IL1B mRNA in macrophages.
PAMPs, components of bacteria and viruses that are recognized by pattern recognition receptors such as TLRs, include cell wall components, LPS, lipoteichoic acid, flagella, and fimbriae. Ligand binding by TLRs induces the expression of genes involved in immune and inflammatory responses (27). The genome of S. enterica serovar Typhimurium contains 13 operons with homology to fimbrial genes. Tükel et al. reported that CsgA, a major subunit of thin curled fimbriae, is recognized by TLR5 (42). There are many reports that Porphyromonas gingivalis fimbriae are potent stimulators of immune and proinflammatory responses (4346). Detection of P. gingivalis FimA, a major fimbrial protein, by TLR2 results in the activation of macrophages or epithelial cells (34, 35). However, our result showed that Salmonella FimA did not induce the expression of proinflammatory cytokines in macrophages. Salmonella FimA and P. gingivalis FimA are both major protein components of the fimbriae. However, their amino acid sequences differ greatly, which could be the main reason for the differences in their activities.
Type 1 fimbriae are proteinaceous filamentous structures that are present on the surface of many members of the Enterobacteriaceae, including S. enterica and E. coli (13, 14). They are composed mainly of FimA and FimH proteins, which are located at the tip of the fimbrial shaft and mediate mannose-sensitive bacterial adhesion to target cells (1517). Comparison of fim gene clusters from S. enterica serovar Typhimurium and E. coli revealed that they differ substantially in gene organization and composition (47). E. coli FimH is reported to be a protein ligand for TLR4 (25, 26). In our study, Salmonella FimH showed an effect on the induction of expression of proinflammatory cytokines via TLR4, even though its amino acid sequence homology with E. coli FimH is only 15%. TLR4 plays an important role as an LPS receptor (32, 48). Effective recognition of LPS by TLR4 requires the assembly of a signaling complex comprised of LPS-binding protein, CD14, and the activating protein MD-2. These accessory components act in concert to bring LPS to TLR4 to induce the subsequent signaling response (29, 40, 41). Mossman et al. showed that E. coli FimH, unlike LPS, does not require MD-2 and CD14 for the expression of its TLR4-mediated activity (26). However, our results showed that these factors are necessary for the induction of TLR4-mediated activity by Salmonella FimH. This difference in activity could be due to differences in amino acid sequence (low amino acid sequence homology) between FimH proteins from Salmonella and E. coli.
E. coli FimH has been crystallized, and the mannose-binding lectin domain was mapped to the N terminus of the molecule, whereas the domain that comprises the fimbrial shaft was mapped to the C terminus of the FimH molecule (49). In silico simulations showed that even with the limited identity of their primary sequences, FimH proteins from E. coli and Salmonella share remarkably similar complex receptor-binding functions and structural properties (50). Our results demonstrated that the N terminus of Salmonella FimH is likely more important than its C terminus for the expression of its activity. We therefore attempted to create a recombinant protein consisting of amino acids 1 to 161 of FimH, without its signal sequence, but did not succeed. In the future, we hope to determine the structure required for FimH activity. Subsequently, we eliminated the possibility that TLR4-mediated FimH activity could be an artifact of LPS contamination of purified FimH by showing (i) that, unlike LPS, the ability of FimH to induce IL1B expression is not thermostable and (ii) that polymyxin B did not affect FimH-induced IL1B expression. These results strongly suggest that TLR4-mediated FimH activity is not due to LPS contamination.
In subsequent experiments, we focused on the signal transduction pathways governing FimH-induced expression of IL1B in macrophages. IL-1β, TNF-α, and IL-6 are proinflammatory cytokines that are mainly secreted by macrophages and have multiple functions in regulating immune responses (51). The cellular processes that lead to the expression of inflammatory cytokines are regulated mainly by the MAPK and NF-κB signaling pathways (52). MAPKs, including ERK, JNK, and p38, are an important group of serine/threonine kinases that transduce a variety of extracellular stimuli through a signaling cascade of protein phosphorylation leading to transcription factor activation (53). NF-κB, a key transcription factor comprised of p50 and p65 subunits (54), makes complexes with IκB in the cytosol of unstimulated cells. Upon stimulation, IκB is phosphorylated by the activated cellular IκB kinase complex, triggering its degradation and resulting in the translocation of NF-κB into the nucleus to promote the transcription of target genes (3638). In this study, we showed that, in addition to the activation of the NF-κB pathway, FimH induces the phosphorylation of ERK, JNK, and p38 MAPK in macrophages. Furthermore, our results showed that inhibition of NF-κB and p38 MAPK blocked FimH-induced IL1B expression, indicating the involvement of both NF-κB and p38 MAPK signaling pathways in this process.
During the establishment of Salmonella infection, SPI-1 and SPI-2 play crucial roles in the invasion of host cells and intracellular survival, respectively (47). SPI-1 is also implicated in the onset of inflammatory diarrhea via a mechanism involving modulation of inflammatory responses in enteric tissue (810). Moreover, Salmonella flagellin, a component of the flagellar filament, induces inflammatory responses via TLR5 in epithelial cells (33, 55, 56). These events are considered important for intestinal colonization by Salmonella (11, 12). Therefore, the expression of FimH-induced proinflammatory cytokines via TLR4 might also play a significant role in the pathogenicity of Salmonella.
In conclusion, type 1 fimbriae are involved in the expression of proinflammatory cytokines in macrophages infected with S. enterica serovar Typhimurium. Furthermore, FimH, but not FimA, is a PAMP that is recognized by TLR4 in the presence of MD-2 and CD14 and plays a significant role in the expression of proinflammatory cytokines in macrophages. Thus, the effect of FimH function on the pathogenicity of Salmonella needs to be further investigated in the future.

MATERIALS AND METHODS

Reagents.

Reagents for cell culture, LPS (E. coli O111:B4), and polymyxin B were purchased from Sigma-Aldrich (St. Louis, MO). SB203580, SP600125, PD98059, MG-132, and TAK-242 were obtained from Millipore (Billerica, MA) and were dissolved in dimethyl sulfoxide (DMSO). When these drugs were used, the final concentration of DMSO in the culture medium was 0.1%; this concentration of solvent did not affect cell responses.

Bacterial strains, plasmids, and growth conditions.

The bacterial strains used in this study were derived from wild-type S. enterica serovar Typhimurium strain 14028s. Deletion mutants of the fimA and fimH genes were constructed using the Red recombination system (57). To delete the fimA 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 fimA gene (5′-ATGAAACATAAATTAATGACCTCTACTATTGCGAGTCTGATGTTTGTGTAGGCTGGAGCTGCTTC-3′ and 5′-TTATTCGTATTTCATGATAAAGGTGGCGTCGGCATTAGCCTGGCCCATATGAATATCCTCCTTAGT-3′). Kanamycin-resistant strains were obtained by transforming the PCR products into strain 14028s harboring the λ Red recombinase on plasmid pKD46. Disruption of the fimA gene was confirmed by PCR using fimA gene-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 fimA gene. The fimH gene was deleted in the same way but using primers 5′-CTGGCGACGGTTTGCCGTAATTCAAACGGGACGGCGACCGATATCGTGTAGGCTGGAGCTGCTTC-3′ and 5′-AATCGACTCGTAGATAGCCGCGCGCAGTAAACGGCCCTTCCGCCGCATATGAATATCCTCCTTAGT-3′. Bacteria were grown at 37°C in Luria broth (LB). Kanamycin was used at 50 μg/ml.

Purification of recombinant proteins.

The S. enterica serovar Typhimurium strain 14028s fimA gene (492-bp DNA fragment, except for its 66-bp signal sequence), fimH gene (942-bp DNA fragment, except for its 66-bp signal sequence), and fimH gene (459-bp DNA fragment from positions 550 to 1008) carrying a deletion of its N-terminal region were each cloned into the pET151/D-TOPO vector using Champion pET directional TOPO expression kits (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. The following DNA fragments were amplified by PCR using the corresponding primers: fimA (5′-CACCATGGCTGATCCTACTCCGGTGAGCGTG-3′ and 5′-TTATTCGTATTTCATGATAAAGGTG-3′) (492-bp fragment), fimH (5′-CACCACGGTTTGCCGTAATTCAAAC-3′ and 5′-TTAATCATAATCGACTCGTAGATAG-3′) (942-bp fragment), and the fimH C-terminal region (5′-CACCGTATATACCATTAGCTACAGC-3′ and 5′-TTAATCATAATCGACTCGTAGATAG-3′) (459-bp fragment). The resulting constructs were verified by direct sequencing. E. coli BL21(DE3) cells transformed with expression vectors containing each gene were grown at 37°C in 100 ml of LB medium containing ampicillin (50 μg/ml). When the optical density of the culture reached 0.5 at 600 nm, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0 mM to induce protein expression. Bacterial growth continued for another 12 h at 26°C. The recombinant proteins were then purified under denaturing conditions using Ni-nitrilotriacetic acid (NTA) affinity chromatography (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Purified protein concentrations were determined using a Quick Start Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as the standard. The LPS content of recombinant protein was determined using a Limulus ES-II single test (Wako Pure Chemical Corp., Osaka, Japan) according to the manufacturer’s instructions.

Cell culture and bacterial infection.

J774 E, a mannose receptor-positive murine macrophage cell line, and HEK293 cells were maintained at 37°C in an incubator with a 5% CO2 atmosphere. Cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (HyClone, Logan, UT), 100 U/ml penicillin, and 100 μg/ml streptomycin. The day before infection, macrophages were plated at a density of 0.5 × 106 cells/well in 12-well tissue culture plates (Falcon, Franklin Lakes, NJ) in medium without antibiotics. Bacteria (approximately 5 × 106) were added, and the plates were centrifuged at 500 × g for 10 min at room temperature. The number of bacteria added to cells was determined by plating the diluted bacteria onto LB agar plates. The cells were then incubated for 20 min at 37°C to permit phagocytosis of the bacteria, and free bacteria were removed by three washes with phosphate-buffered saline (PBS). After washing, Dulbecco’s modified Eagle’s medium containing 12 μg/ml gentamicin was added to kill extracellular bacteria, after which the cells were incubated at 37°C for the indicated times. To examine the number of bacteria taken up by macrophages, a solution of 0.1% Triton X-100 was added to each well to lyse cells after washing with PBS, and the number of bacteria was determined by plating the diluted bacteria onto LB agar plates.

RNA preparation and quantitative real-time PCR.

Total RNA was extracted from macrophages in 12-well plates using TRIzol reagent (Life Technologies) according to the manufacturer’s protocols and incubated with DNase I (Life Technologies). First-strand cDNA was synthesized using a High-Capacity RNA-to-cDNA kit (Life Technologies) according to the manufacturer’s protocol. qRT-PCR was performed in 20-μl reaction mixtures containing 1 μl of cDNA, 0.5 μM each primer, and 10 μl of PowerUp SYBR green master mix (Life Technologies). Amplification was carried out in 96-well optical plates on a 7300 real-time PCR system (Life Technologies) with an initial incubation step for 2 min at 50°C, followed by 2 min at 95°C and then 40 cycles of 95°C for 15 s and 60°C for 1 min. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as an internal standard for quantification of the qRT-PCR products. Threshold cycle values were calculated from the amplification plots, and the relative expression level of each gene in stimulated cells was compared with that in unstimulated cells after both values were normalized according to GAPDH expression. Each sample was analyzed in triplicate. The sequences of primers were as follows: 5′-TGTCTTGGCCGAGGACTAAGG-3′ and 5′-TGGGCTGGACTGTTTCTAATGC-3′ for IL1B, 5′-CCAAACTGGATATAATCAGGAAAT-3′ and 5′-CTAGGTTTGCCGAGTAGATCTC-3′ for IL6, 5′-AGAAACACAAGATGCTGGGACAGT-3′ and 5′-CCTTTGCAGAACTCAGGAATGG-3′ for TNFA, and 5′-TGGCCAAGGTCATCCATGACAAC-3′ and 5′-TCCAGAGGGGCCATCCACAGTCTTCTG-3′ for GAPDH.

Western blot analysis.

Western blot analyses were performed essentially as described previously (58). Cell lysates from macrophages were prepared, and detection of ERK, p38, and JNK expression was performed as described previously (59). IκB-α was detected using a 1:500 dilution of anti-IκB-α antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Luciferase assay.

HEK293 cells, HEK293 cells expressing mouse TLR4 (HEK293/mTLR4), and HEK293 cells expressing mouse TLR4 and MD-2/CD14 (HEK293/mTLR4/MD-2/CD14) were purchased from InvivoGen (San Diego, CA). These cells were transiently transfected with the pNiFty-Luc NF-κB firefly luciferase reporter construct (InvivoGen) and the pRL-SV40 Renilla luciferase reporter vector (Promega, Madison, WI) as a control for transfection efficiency using Lipofectamine 3000 reagent (Life Technologies). Twenty-four hours after transfection, cells were stimulated with or without an agonist. Six hours later, cell lysates were harvested, and luciferase activity was measured using the Dual-Glo luciferase assay system (Promega) according to the manufacturer’s instructions. All luciferase assays were performed with triplicate samples. NF-κB firefly luciferase levels were normalized to internal Renilla luciferase levels as a transfection control.

Statistical analysis.

Each experiment was performed at least three times. The results are expressed as the means ± standard deviations. Data were analyzed by one-way analysis of variance followed by Tukey’s multiple-comparison test. Differences at a P value of <0.05 were considered statistically significant.

ACKNOWLEDGMENTS

We thank Saki Isono, Misa Yoshimura, Tomoyo Shimizu, and Takahiro Takagi for their technical assistance.
This work was supported by JSPS KAKENHI grant number 15K08049 (to K.U.).

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Information & Contributors

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Published In

cover image Infection and Immunity
Infection and Immunity
Volume 87Number 3March 2019
eLocator: 10.1128/iai.00881-18
Editor: Manuela Raffatellu, University of California San Diego School of Medicine

History

Received: 14 December 2018
Accepted: 14 December 2018
Published online: 21 February 2019

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Keywords

  1. FimH
  2. Salmonella enterica serovar Typhimurium
  3. Toll-like receptor 4
  4. type 1 fimbriae
  5. proinflammatory cytokines

Contributors

Authors

Kei-ichi Uchiya
Department of Microbiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan
Yurie Kamimura
Department of Microbiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan
Ayumi Jusakon
Department of Microbiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan
Toshiaki Nikai
Department of Microbiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan

Editor

Manuela Raffatellu
Editor
University of California San Diego School of Medicine

Notes

Address correspondence to Kei-ichi Uchiya, [email protected].

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