TLR5-mediated cell activation by LM
Previous studies have demonstrated that the expression of flagellin in LM is temperature dependent. Flagella are present, as detected by electron microscopy, and the structural gene for flagellin, flaA, is transcribed maximally after growth at 22°C or below, but expression is repressed at 37°C (Peel et al., 1988; Dons et al., 1992). Downregulation of flagellin by LM at 37°C may represent a mechanism by which the bacterium attempts to evade TLR5-mediated host immunity. To test this hypothesis, we examined the temperature-dependent regulation of flagellin expression in a larger panel of LM wild-type strains. Six well-characterized laboratory-adapted strains of LM were tested for the ability to activate TLR5-transfected HeLa cells after growth at 22°C or 37°C (Table 1, Fig. 1A). Although all LM strains tested could activate cells through TLR5 after growth at 22°C, surprisingly, one LM strain (10403s) retained the ability to activate cells through TLR5 after growth at 37°C.
Table 1. . TLR5-mediated cell activation for LM laboratory-adapted strains and clinical isolates after growth at 22°C or 37°C.
|Strain||Source||Fold induction of NF-κB through TLR5 after growth at|
| 10403s||Faeces (rabbit)||12.6a||6.4a|
| ScottA||Human outbreak||13.2a||1.3|
| 43251||Mesenteric LN|| 9.9a||0.8|
| EGD||Human infection||10.9a||1.1|
| LO28||Faeces (human)||11.8a||1.2|
| U14||Blood|| 9.9a||1.0|
| U17||Blood|| 9.5a||0.8|
Figure 1. TLR5-mediated cell activation and flagellin expression by LM strains. A. Induction of NF-κB by live LM (1.0 × 107 cfus) in HeLa cells transfected with empty vector or TLR5. For stimulations, LM was grown at either 22°C or 37°C as indicated. This figure demonstrates cell activation through TLR5 for four representative strains tested (10403s and DOH 2672 activate cells through TLR5 after growth at 37°C; 43251 and DOH 2873 do not activate cells through TLR5 at 37°C). Relative light units represent the mean ratio of ELAM-Luc/β-actin Renilla-Luc activity in triplicate wells. Results are representative of two independent experiments. Bar, one standard deviation. B. RT-PCR demonstrating the expression of flaA or the control gene, hly, by LM in the spleens isolated ex vivo 3 days after intravenous infection with 1.0 × 105 cfus of either 10403s or 43251. Data are shown for two out of three representative mice infected with LM strain 10403s, and two out of four representative mice infected with strain 43251. C. Induction of NF-κB by serial dilutions of live LM [1.0 × 107 cfus (1:1) to 3.9 × 104 (1:256)] strain 10403s in HeLa cells transfected with empty vector, TLR5 or TLR2. Relative light units represent the mean ratio of ELAM-Luc/β-actin Renilla-Luc activity in four duplicate wells. Results are representative of two independent experiments. Bar, one standard deviation. D. Induction of NF-κB by IL-1, purified S. typhimurium flagellin or the indicated LM (1.0 × 107 cfus) strains in HeLa cells transfected with empty vector, TLR5 or TLR2. Relative light units represent the mean ratio of ELAM-Luc/β-actin Renilla-Luc activity in four duplicate wells. Results are representative of two independent experiments. Bar, one standard deviation. ΔflaA, flagellin-deficient LM; pflaA, plasmid encoding full-length LM flaA.
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To determine whether TLR5-mediated cell activation by LM 10403s grown at 37°C is unique to this laboratory-adapted strain, we examined TLR5-mediated cell activation in a panel of 25 LM clinical isolates obtained from sterile site patient cultures (Table 1, Fig. 1A). Similar to the laboratory-adapted strains, all LM clinical isolates activated cells through TLR5 after growth at 22°C. However, at 37°C, only five out of 25 clinical isolates (20%) retained the ability to activate cells through TLR5. Furthermore, all LM strains that activated cells through TLR5 were also motile at 37°C in soft agar. These results indicate that ≈ 20% of naturally occurring pathogenic LM strains retained flagella expression and the ability to activate cells through TLR5 after growth at 37°C in vitro.
Flagellin expression by LM
In LM, there is a direct correlation between transcription of the flagellin structural gene, flaA, and the presence of flagellin protein (Peel et al., 1988; Dons et al., 1992). Thus, to validate the fact that TLR5-mediated cell activation resulted from flagellin expression, the presence of flaA mRNA was examined for LM. Two clinical isolates and one laboratory-adapted LM strain that activated cells through TLR5 after growth at 37°C, and four clinical isolates and two laboratory-adapted LM strains that did not, were examined (Table 2). Although all LM strains expressed the flaA gene after growth at 22°C, only strains that could mediate cell activation through TLR5 after growth at 37°C expressed the flaA gene. In contrast, for all LM strains that did not activate cells through TLR5 after growth at 37°C, flaA transcript could not be detected.
Table 2. . RNA expression of the LM flagellin structural gene, flaA, in LM strains after growth in vitro at either 22°C or 37°C as determined by RT-PCR.
| ||flaA gene expression at|
|Activates cells through|
|TLR5 after growth at 37°C|
|Does not activate cells through|
|TLR5 after growth at 37°C|
As our primary interest was to examine the contribution of LM flagellin expression during infection, we picked two well-characterized laboratory-adapted LM strains [one that expressed flaA and retained the ability to activate cells through TLR5 at 37°C (10403s) and one that did not (43251)] to examine whether the distinct differences in flagellin expression and TLR5-mediated cell activation for these strains would extend to similar differences during in vivo infection. This was performed by examining the expression of flaA by reverse transcription polymerase chain reaction (RT-PCR) in LM-infected spleens directly ex vivo from mice infected with LM strain 10403s or 43251. Although flaA mRNA was consistently expressed in LM recovered from mice infected with 10403s, it could not be detected in LM recovered from mice infected with 43251 (Fig. 1B). As a positive control for LM RNA isolation from infected tissue, hly mRNA, the expression of which is upregulated by LM during infection, was uniformly detected in tissue from mice infected with either 10403s or 43251. Thus, the expression of flaA during in vivo infection by LM strains 10403s and 43251 is identical to the pattern of flaA expression and TLR5-mediated cell activation by these strains when grown at 37°C in vitro. These data demonstrate that the difference in flagellin expression at 37°C in vitro between LM strains 10403s and 43251 extends directly to differences during in vivo infection.
To quantify better the relative ability of LM to activate cells through TLR5 after growth at 37°C compared with 22°C, the degree of activation for LM 10403s grown at 37°C was compared with serial twofold dilutions of LM 10403s grown at 22°C (Fig. 1C). The induction of NF-κB through TLR5 for LM grown at 22°C was consistently higher than for LM grown at 37°C in undiluted samples, and became comparable only after five to six serial twofold dilutions [relative amount: 1/64–1/32 (2–3%)]. In contrast, the induction of NF-κB through TLR2 for LM grown at 37°C was similar to that for LM grown at 22°C. These data demonstrate that, although some LM strains retained the ability to activate cells through TLR5 at 37°C, the relative ability to activate cells through TLR5 after in vitro growth at 37°C is considerably less than that at 22°C.
Pathogenesis of flagellin-deficient LM
As only ≈ 20% of LM strains causing invasive human infection retained the ability to activate cells through TLR5 after growth at 37°C, this suggests that flagellin expression in vivo and at 37°C does not play a critical role in LM pathogenesis. To address this prediction, we generated a flagellin-deficient LM strain (LM ΔflaA) by targeted disruption of the flaA gene (Shetron-Rama et al., 2003) and compared the virulence after intravenous inoculation of mice with flagellin-deficient and wild-type LM. This mutant was constructed in LM strain 10403s, which expressed flagellin and activated cells through TLR5 under all conditions tested. As predicted, this mutant was unable to activate cells via TLR5 at either 37°C or 22°C, although the defect in TLR5-mediated cell activation for this mutant could be fully restored by complementation in trans with a construct containing the full-length flaA gene (Fig. 1D).
As TLR5 is one of at least three known TLRs (TLR2 for LM peptidoglycan and TLR9 for bacterial CpG DNA) capable of recognizing PAMPs present in LM, we examined the relative contribution of LM flagellin to proinflammatory cytokine production in primary splenocytes. In splenocytes from normal mice, an ≈ 50% reduction in tumour necrosis factor (TNF)-α-producing CD11b+ splenocyte cells was observed after stimulation with 10403s ΔflaA compared with wild-type 10403s (Fig. 2). TNF-α production in splenocytes is dependent on the TLR/MyD88 signalling pathway, as the response to both 10403s and 10403s ΔflaA was substantially reduced in splenocytes from MyD88-deficient mice. These data demonstrate that LM flagellin contributes to, but is not the only signal inducing, proinflammatory cytokine production in primary cells containing multiple TLRs capable of LM recognition.
Figure 2. TNF-α production by splenocytes isolated ex vivo from C57Bl/6 (MyD88+/+) or MyD88-deficient back-crossed six times to C57Bl/6 (MyD88–/–) mice after stimulation with WT LM, flagellin-deficient LM [LM (flaA)] or no stimulation, as determined by intracellular cytokine staining. The mean percentage ± standard error of CD11b+ cells producing TNF-α after stimulation, as determined by intracellular cytokine staining, is indicated (n = four mice per group).
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After intravenous inoculation of C57 H-2bxd mice with 1.0 × 105 cfus of either 10403s ΔflaA or wild-type 10403s (n = 6 per group), 100% of the mice became moribund and were euthanized or died by days 3–5 after infection. After infection with an 80% lower inoculum, 2.0 × 104 cfus of 10403s ΔflaA or wild-type 10403s, no mortality was observed (n > 20 per group). These data are consistent with the previously defined LD50 (104.7−105 cfus) after intravenous inoculation for LM strain 10403s in C57Bl/6 mice (White et al., 2000; Shedlock et al., 2003). Although no mortality was observed after intravenous infection with 2.0 × 104 cfus of either LM strain 10403s ΔflaA or wild-type 10403s, bacterial replication occurred in vivo as determined by the numbers of LM recovered from the spleen and liver beginning at day 3 after infection compared with the initial inoculum (Fig. 3). For mice infected with either wild-type 10403s or 10403s ΔflaA, reduced numbers of LM were present by day 7 after infection, indicating that bacterial clearance had occurred. However, at each of these time points, there were no differences in the number of LM recovered from mice infected with 10403s ΔflaA compared with wild-type 10403s (Fig. 3). Thus, LM flagellin does not affect bacterial replication or clearance after intravenous inoculation.
Figure 3. Numbers of LM recovered from the spleen or liver of mice inoculated intravenously with 2.0 × 104 cfus of either 10403s or 10403s ΔflaA. Each data point indicates the log10 cfu from an individual mouse. Data presented represent the cfus in mice pooled from at least two independent experiments that independently yielded similar results. Bar, geometric mean.
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While these findings indicate that flagellin does not affect pathogenesis after intravenous challenge, it is possible that flagellin may do so in the context of infection acquired orally. In polarized intestinal epithelial cells, TLR5 is expressed only on the basolateral surface and is excluded from the luminal surface (Gerwitz et al., 2001a,b). This pattern of TLR5 expression in intestinal epithelial cells is hypothesized to limit inflammation to endogenous gut flora, allowing inflammation and recruitment of professional APCs only in response to invasive flagellated bacteria able to transverse the intestinal epithelial cell barrier (Sierro et al., 2001). Thus, the contribution of TLR5 in immunity to LM infection may vary with the route of experimental infection.
LM inoculated by orogastric gavage are invasive and cause bacteraemia (Huleatt et al., 2001; Pope et al., 2001; Kursar et al., 2002). The kinetics of bacterial replication and clearance, and the generation of LM-specific T cells after intravenous and oral infection are similar, although the oral dose required to elicit an intravenous equivalent response is significantly greater (oral 109 versus intravenous 104 cfus) (Huleatt et al., 2001; Pope et al., 2001). After orogastric infection, 33% of mice infected with 10403s ΔflaA (n = 12) and 11% of mice infected with wild-type 10403s (n = 9) became moribund and were sacrificed or died. This difference in survival did not reach statistical significance (P = 0.29). In the remaining mice, a similar ability to translocate across the gastrointestinal epithelium and disseminate to the spleen, mesenteric lymph nodes and liver was observed for 10403s ΔflaA and wild-type 10403s, as determined by the number of LM cfus by day 3 after infection (Fig. 4). Furthermore, both 10403s ΔflaA and wild-type 10403s were substantially cleared from these same tissues by day 7.
Figure 4. Numbers of LM recovered from the spleen, mesenteric lymph nodes or liver of mice inoculated by the orogastric route with 1.0 × 109 cfus of either 10403s or 10403s ΔflaA. Each data point indicates the log10 cfu from an individual mouse. Data represent the cfus in mice pooled from at least two independent experiments that yielded similar results. Bar, geometric mean. Day 7 cfus spleen: 10403s versus 10403s ΔflaA, P = 0.08; mesenteric lymph nodes: 10403s versus 10403s ΔflaA, P = 0.49; liver: 10403s versus 10403s ΔflaA, P = 0.49 (Mann–Whitney test).
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Adaptive immunity triggered by flagellin-deficient LM
As exogenously added flagellin is able to enhance the T-cell response to other bacterial pathogens (McSorley et al., 2002), the contribution of flagellin to the generation of adaptive immune responses to LM was examined (Fig. 5). After intravenous or oral infection with either 10403s ΔflaA or wild-type 10403s, no significant differences were observed in either the percentage or the total number of CD8 or CD4 T cells specific for the LM major histocompatibility (MHC) class I (LLO 91–99) or class II (LLO 189–201) immunodominant peptides. These data demonstrate that, during infection, LM flagellin does not substantially alter the generation of adaptive immune responses, and are consistent with the similar kinetics of bacterial clearance observed between days 3 and 7 after infection. These results are consistent with the generation of normal numbers of LM-specific CD8 T cells and only a modest decline in LM-specific CD4 T cells in MyD88-deficient mice (which lack functional TLR5 and TLR2) (Way et al., 2003). These findings also suggest that LM flagellin does not alter the generation of LM-specific T cells through non-TLR-mediated pathways.
Figure 5. Enumeration of LM-specific CD8 and CD4 T cells. A. Percentage of IFN-γ-positive splenic CD8 or CD4 T cells on day 7 after infection with 10403s or 10403s ΔflaA after stimulation with the LM MHC class I-restricted peptide LLO 91–99 (CD8 T cells), MHC class II-restricted peptide LLO 189–201 (CD4 T cells) or no peptide. Top and bottom, the percentage of LM-specific T cells after intravenous and orogastric infection respectively. B. Total numbers of IFN-γ-producing CD8 and CD4 T cells per mouse spleen after stimulation with the indicated MHC class I- or class II-restricted LM-specific peptides. These data represent 6–12 mice per group combined from two independent experiments that yielded similar results. Bar, standard error. Intravenous infection: CD8 T cells 10403s versus 10403s ΔflaA, P = 0.22; CD4 T cells 10403s versus 10403s ΔflaA, P = 0.26. Oral infection: CD8 T cells 10403s versus 10403s ΔflaA, P = 0.47; CD4 T cells 10403s versus 10403s ΔflaA, P = 0.47 (Mann–Whitney test).
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In summary, the data presented in this study demonstrate that, although all LM strains are able to activate cells through TLR5 when grown at 22°C, only ≈ 20% of LM strains retain flagellin expression at 37°C in vitro and perhaps in vivo. Although less flagellin is produced by these LM strains after growth at 37°C compared with 22°C, these strains retained the ability to activate cells though TLR5. When an LM mutant with targeted flagellin deficiency was generated and compared with a parental LM strain expressing flagellin, no significant differences were observed in bacterial replication, bacterial clearance or induction of LM-specific T cells after either intravenous or oral infection. These results indicate that flagellin is neither an essential trigger for innate or adaptive immunity to LM, nor does it exact a physiologically important metabolic penalty, the relief from which (following downregulation at temperatures present in mammals in vivo) allows LM to invade or to replicate more efficiently in vivo. In the context of the extreme susceptibility of MyD88-deficient mice, and lack of susceptibility of TLR2-deficient mice, to primary LM infection (Edelson and Unanue, 2002; Seki et al., 2002), these findings suggest that functional redundancy exists between known TLRs (pattern recognition receptors) in innate immunity to LM infection. Alternatively, the lack of a significant phenotype for flagellin-deficient compared with flagellin-expressing wild-type LM may reflect the relatively low amount of flagellin produced by LM at 37°C, compared with that produced at 22°C. This alternative would support the hypothesis that LM downregulates flagellin as a means of evading host immunity through TLR5.