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Vibrio cholerae proteome-wide screen for immunostimulatory proteins identifies phosphatidylserine decarboxylase as a novel Toll-like receptor 4 agonist.

Thanawastien A, Montor WR, Labaer J, Mekalanos JJ, Yoon SS - PLoS Pathog. (2009)

Bottom Line: An enzymatically inactive PSD mutant and heat-inactivated PSD induced approximately 40% and approximately 15% of IL-6 production compared to that by native PSD, respectively.Anti-BSA response was decreased in TLR4-deficient mice immunized with BSA in combination with PSD, further proving the role of TLR4 in PSD signaling in vivo.Taken together, these results provide evidence for the identification of V. cholerae PSD as a novel TLR4 agonist and further demonstrate the potential application of PSD as a vaccine adjuvant.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA.

ABSTRACT
Recognition of conserved bacterial components provides immediate and efficient immune responses and plays a critical role in triggering antigen-specific adaptive immunity. To date, most microbial components that are detected by host innate immune system are non-proteinaceous structural components. In order to identify novel bacterial immunostimulatory proteins, we developed a new high-throughput approach called "EPSIA", Expressed Protein Screen for Immune Activators. Out of 3,882 Vibrio cholerae proteins, we identified phosphatidylserine decarboxylase (PSD) as a conserved bacterial protein capable of activating host innate immunity. PSD in concentrations as low as 100 ng/ml stimulated RAW264.7 murine macrophage cells and primary peritoneal macrophage cells to secrete TNFalpha and IL-6, respectively. PSD-induced proinflammatory response was dependent on the presence of MyD88, a known adaptor molecule for innate immune response. An enzymatically inactive PSD mutant and heat-inactivated PSD induced approximately 40% and approximately 15% of IL-6 production compared to that by native PSD, respectively. This suggests that PSD induces the production of IL-6, in part, via its enzymatic activity. Subsequent receptor screening determined TLR4 as a receptor mediating the PSD-induced proinflammatory response. Moreover, no detectable IL-6 was produced in TLR4-deficient mouse macrophages by PSD. PSD also exhibited a strong adjuvant activity against a co-administered antigen, BSA. Anti-BSA response was decreased in TLR4-deficient mice immunized with BSA in combination with PSD, further proving the role of TLR4 in PSD signaling in vivo. Taken together, these results provide evidence for the identification of V. cholerae PSD as a novel TLR4 agonist and further demonstrate the potential application of PSD as a vaccine adjuvant.

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Stimulatory effect of PSD on mouse TLR4.(A) SEAP reporter activity was measured in HEK293 cells transfected with each mouse TLR construct (indicated at the bottom) and plotted as a 3-D bar graph. Cells expressing each TLR were treated with its own control ligand (blue bars), WT-PSD (3 µg/ml, red bars), VC0222 (3 µg/ml, yellow bars) and PBS (gray bars) in two independent cultures. Average activity values were used for plotting. LPS levels in the final preparation in PSD and VC0222 were 0.035 (±0.00191) and 0.027 (±0.0007) EU/ml, respectively. (B) Peritoneal macrophage cells isolated from C3H/HeOuJ (TLR4 WT) or C3H/HeJ (TLR4-deficient) mice were treated with S. typhimurium LPS (100 ng/ml), WT-PSD (1.5 µg/ml), VC0222 (1.5 µg/ml) and PBS for 9 hrs and IL-6 level in each treatment was measured after 3 (gray bars), 6 (black bars) and 9 hrs (hatched bars). LPS levels in the final preparation in PSD and VC0222 were 0.035 (±0.00191) and 0.027 (±0.0007) EU/ml, respectively. *p<0.01 vs. IL-6 production from TLR4 intact macrophages in each time point.
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ppat-1000556-g005: Stimulatory effect of PSD on mouse TLR4.(A) SEAP reporter activity was measured in HEK293 cells transfected with each mouse TLR construct (indicated at the bottom) and plotted as a 3-D bar graph. Cells expressing each TLR were treated with its own control ligand (blue bars), WT-PSD (3 µg/ml, red bars), VC0222 (3 µg/ml, yellow bars) and PBS (gray bars) in two independent cultures. Average activity values were used for plotting. LPS levels in the final preparation in PSD and VC0222 were 0.035 (±0.00191) and 0.027 (±0.0007) EU/ml, respectively. (B) Peritoneal macrophage cells isolated from C3H/HeOuJ (TLR4 WT) or C3H/HeJ (TLR4-deficient) mice were treated with S. typhimurium LPS (100 ng/ml), WT-PSD (1.5 µg/ml), VC0222 (1.5 µg/ml) and PBS for 9 hrs and IL-6 level in each treatment was measured after 3 (gray bars), 6 (black bars) and 9 hrs (hatched bars). LPS levels in the final preparation in PSD and VC0222 were 0.035 (±0.00191) and 0.027 (±0.0007) EU/ml, respectively. *p<0.01 vs. IL-6 production from TLR4 intact macrophages in each time point.

Mentions: Our results shown in Fig. 3A indicated that V. cholerae PSD elicits proinflammatory responses in a MyD88-dependent manner. Since most TLRs use MyD88 as a key adaptor protein to recruit downstream signaling molecules [1],[31], we hypothesized that PSD may signal through a TLR to activate innate immune responses and the production of proinflammatory cytokines. To test this hypothesis, HEK293 (human embryonic kidney) cells, which do not express endogenous TLR [36], were transfected with each of the individual TLRs (TLR2, 3, 4, 5, 7, and 9) and assayed for activation by PSD. To ensure full responsiveness to LPS, the plasmid expressing tlr4 co-transcribes genes encoding CD14 and MD2, which are involved in LPS responses [37]. HEK293 cells were also transfected with a reporter construct in which the expression of secreted alkaline phosphatase (SEAP) is driven from an NF-κB inducible promoter. Appropriate positive controls (Fig. S2) were tested to compare their NF-κB signaling activity with that of PSD. As shown in Fig. 5A, HEK293 cells expressing each TLR responded to its corresponding ligand (blue bars). No cross reactivity was detected in HEK293 cells responding to other control ligands (data not shown). We observed that PSD-induced NF-κB activation was most efficiently detected in HEK293 cells expressing TLR4/MD2/CD14 in comparison to the other TLRs, indicating that PSD likely signals through TLR4 (Fig. 5A).


Vibrio cholerae proteome-wide screen for immunostimulatory proteins identifies phosphatidylserine decarboxylase as a novel Toll-like receptor 4 agonist.

Thanawastien A, Montor WR, Labaer J, Mekalanos JJ, Yoon SS - PLoS Pathog. (2009)

Stimulatory effect of PSD on mouse TLR4.(A) SEAP reporter activity was measured in HEK293 cells transfected with each mouse TLR construct (indicated at the bottom) and plotted as a 3-D bar graph. Cells expressing each TLR were treated with its own control ligand (blue bars), WT-PSD (3 µg/ml, red bars), VC0222 (3 µg/ml, yellow bars) and PBS (gray bars) in two independent cultures. Average activity values were used for plotting. LPS levels in the final preparation in PSD and VC0222 were 0.035 (±0.00191) and 0.027 (±0.0007) EU/ml, respectively. (B) Peritoneal macrophage cells isolated from C3H/HeOuJ (TLR4 WT) or C3H/HeJ (TLR4-deficient) mice were treated with S. typhimurium LPS (100 ng/ml), WT-PSD (1.5 µg/ml), VC0222 (1.5 µg/ml) and PBS for 9 hrs and IL-6 level in each treatment was measured after 3 (gray bars), 6 (black bars) and 9 hrs (hatched bars). LPS levels in the final preparation in PSD and VC0222 were 0.035 (±0.00191) and 0.027 (±0.0007) EU/ml, respectively. *p<0.01 vs. IL-6 production from TLR4 intact macrophages in each time point.
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Related In: Results  -  Collection

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ppat-1000556-g005: Stimulatory effect of PSD on mouse TLR4.(A) SEAP reporter activity was measured in HEK293 cells transfected with each mouse TLR construct (indicated at the bottom) and plotted as a 3-D bar graph. Cells expressing each TLR were treated with its own control ligand (blue bars), WT-PSD (3 µg/ml, red bars), VC0222 (3 µg/ml, yellow bars) and PBS (gray bars) in two independent cultures. Average activity values were used for plotting. LPS levels in the final preparation in PSD and VC0222 were 0.035 (±0.00191) and 0.027 (±0.0007) EU/ml, respectively. (B) Peritoneal macrophage cells isolated from C3H/HeOuJ (TLR4 WT) or C3H/HeJ (TLR4-deficient) mice were treated with S. typhimurium LPS (100 ng/ml), WT-PSD (1.5 µg/ml), VC0222 (1.5 µg/ml) and PBS for 9 hrs and IL-6 level in each treatment was measured after 3 (gray bars), 6 (black bars) and 9 hrs (hatched bars). LPS levels in the final preparation in PSD and VC0222 were 0.035 (±0.00191) and 0.027 (±0.0007) EU/ml, respectively. *p<0.01 vs. IL-6 production from TLR4 intact macrophages in each time point.
Mentions: Our results shown in Fig. 3A indicated that V. cholerae PSD elicits proinflammatory responses in a MyD88-dependent manner. Since most TLRs use MyD88 as a key adaptor protein to recruit downstream signaling molecules [1],[31], we hypothesized that PSD may signal through a TLR to activate innate immune responses and the production of proinflammatory cytokines. To test this hypothesis, HEK293 (human embryonic kidney) cells, which do not express endogenous TLR [36], were transfected with each of the individual TLRs (TLR2, 3, 4, 5, 7, and 9) and assayed for activation by PSD. To ensure full responsiveness to LPS, the plasmid expressing tlr4 co-transcribes genes encoding CD14 and MD2, which are involved in LPS responses [37]. HEK293 cells were also transfected with a reporter construct in which the expression of secreted alkaline phosphatase (SEAP) is driven from an NF-κB inducible promoter. Appropriate positive controls (Fig. S2) were tested to compare their NF-κB signaling activity with that of PSD. As shown in Fig. 5A, HEK293 cells expressing each TLR responded to its corresponding ligand (blue bars). No cross reactivity was detected in HEK293 cells responding to other control ligands (data not shown). We observed that PSD-induced NF-κB activation was most efficiently detected in HEK293 cells expressing TLR4/MD2/CD14 in comparison to the other TLRs, indicating that PSD likely signals through TLR4 (Fig. 5A).

Bottom Line: An enzymatically inactive PSD mutant and heat-inactivated PSD induced approximately 40% and approximately 15% of IL-6 production compared to that by native PSD, respectively.Anti-BSA response was decreased in TLR4-deficient mice immunized with BSA in combination with PSD, further proving the role of TLR4 in PSD signaling in vivo.Taken together, these results provide evidence for the identification of V. cholerae PSD as a novel TLR4 agonist and further demonstrate the potential application of PSD as a vaccine adjuvant.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA.

ABSTRACT
Recognition of conserved bacterial components provides immediate and efficient immune responses and plays a critical role in triggering antigen-specific adaptive immunity. To date, most microbial components that are detected by host innate immune system are non-proteinaceous structural components. In order to identify novel bacterial immunostimulatory proteins, we developed a new high-throughput approach called "EPSIA", Expressed Protein Screen for Immune Activators. Out of 3,882 Vibrio cholerae proteins, we identified phosphatidylserine decarboxylase (PSD) as a conserved bacterial protein capable of activating host innate immunity. PSD in concentrations as low as 100 ng/ml stimulated RAW264.7 murine macrophage cells and primary peritoneal macrophage cells to secrete TNFalpha and IL-6, respectively. PSD-induced proinflammatory response was dependent on the presence of MyD88, a known adaptor molecule for innate immune response. An enzymatically inactive PSD mutant and heat-inactivated PSD induced approximately 40% and approximately 15% of IL-6 production compared to that by native PSD, respectively. This suggests that PSD induces the production of IL-6, in part, via its enzymatic activity. Subsequent receptor screening determined TLR4 as a receptor mediating the PSD-induced proinflammatory response. Moreover, no detectable IL-6 was produced in TLR4-deficient mouse macrophages by PSD. PSD also exhibited a strong adjuvant activity against a co-administered antigen, BSA. Anti-BSA response was decreased in TLR4-deficient mice immunized with BSA in combination with PSD, further proving the role of TLR4 in PSD signaling in vivo. Taken together, these results provide evidence for the identification of V. cholerae PSD as a novel TLR4 agonist and further demonstrate the potential application of PSD as a vaccine adjuvant.

Show MeSH
Related in: MedlinePlus