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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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PSD-induced proinflammatory response is MyD88-dependent and PSD, in higher concentration, exerts a cytotoxic effect on peritoneal mouse macrophages.(A) IL-6 production in peritoneal macrophages isolated from MyD88+/− (left) or MyD88−/− (right) mouse in response to three purified V. cholerae proteins (PSD, FlaD and VC0222). Cells were treated with the protein in three different final concentrations, 15 µg/ml (black bars), 1.5 µg/ml (gray bars) and 150 ng/ml (hatched bars). Isolation and stimulation of resident murine macrophages were performed as described in Materials and Methods. *p<0.01 vs. IL-6 production in MyD88+/− cells in each corresponding protein concentration. (B) Purity of the protein stimulants was shown in SDS-PAGE. One µg of each protein was separated in 4–12% SDS-PAGE. (C) IL-6 production in peritoneal macrophages isolated from MyD88+/− (left) or MyD88−/− (right) mouse in response to non-protein ligands indicated at the bottom of the graph. Experimental conditions were identical with Fig. 3A. *p<0.01 vs. IL-6 production in MyD88+/− cells in each corresponding protein concentration. (D) LDH activity was measured in the same culture supernatant that was used for IL-6 ELISA. % cytotoxicity was calculated as a relative LDH activity of maximally released LDH by a treatment of 1% triton X-100. *p<0.01 vs. the other treatments.
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ppat-1000556-g003: PSD-induced proinflammatory response is MyD88-dependent and PSD, in higher concentration, exerts a cytotoxic effect on peritoneal mouse macrophages.(A) IL-6 production in peritoneal macrophages isolated from MyD88+/− (left) or MyD88−/− (right) mouse in response to three purified V. cholerae proteins (PSD, FlaD and VC0222). Cells were treated with the protein in three different final concentrations, 15 µg/ml (black bars), 1.5 µg/ml (gray bars) and 150 ng/ml (hatched bars). Isolation and stimulation of resident murine macrophages were performed as described in Materials and Methods. *p<0.01 vs. IL-6 production in MyD88+/− cells in each corresponding protein concentration. (B) Purity of the protein stimulants was shown in SDS-PAGE. One µg of each protein was separated in 4–12% SDS-PAGE. (C) IL-6 production in peritoneal macrophages isolated from MyD88+/− (left) or MyD88−/− (right) mouse in response to non-protein ligands indicated at the bottom of the graph. Experimental conditions were identical with Fig. 3A. *p<0.01 vs. IL-6 production in MyD88+/− cells in each corresponding protein concentration. (D) LDH activity was measured in the same culture supernatant that was used for IL-6 ELISA. % cytotoxicity was calculated as a relative LDH activity of maximally released LDH by a treatment of 1% triton X-100. *p<0.01 vs. the other treatments.

Mentions: To gain an insight into the signaling mechanism(s) by which PSD activates mouse macrophages, PSD was used to stimulate the induction of the proinflammatory cytokine, IL-6, in MyD88−/− (Myeloid Differentiation Factor-88) and MyD88+/− macrophages. MyD88 is one of most commonly used adaptor molecules that mediates signal transduction in mammalian innate immune activation [30],[31]. Upon ligand binding, TLRs recruit many downstream signaling molecules via MyD88 to activate NF-κB, which then transcribes genes involved in the production of proinflammatory cytokines [1]. LPS is a known potent stimulator of MyD88-dependent inflammatory pathway and stimulates a high level of IL-6 secretion from MyD88 positive macrophages compared to MyD88 negative macrophages [32]. When treated with PSD or LPS, MyD88 knockout macrophage cells secreted significantly less IL-6 compared to the level of IL-6 detected in MyD88 positive macrophages (Fig. 3A and C) indicating that PSD triggers through a predominantly MyD88-dependent inflammatory signaling cascade similar to that observed with LPS.


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)

PSD-induced proinflammatory response is MyD88-dependent and PSD, in higher concentration, exerts a cytotoxic effect on peritoneal mouse macrophages.(A) IL-6 production in peritoneal macrophages isolated from MyD88+/− (left) or MyD88−/− (right) mouse in response to three purified V. cholerae proteins (PSD, FlaD and VC0222). Cells were treated with the protein in three different final concentrations, 15 µg/ml (black bars), 1.5 µg/ml (gray bars) and 150 ng/ml (hatched bars). Isolation and stimulation of resident murine macrophages were performed as described in Materials and Methods. *p<0.01 vs. IL-6 production in MyD88+/− cells in each corresponding protein concentration. (B) Purity of the protein stimulants was shown in SDS-PAGE. One µg of each protein was separated in 4–12% SDS-PAGE. (C) IL-6 production in peritoneal macrophages isolated from MyD88+/− (left) or MyD88−/− (right) mouse in response to non-protein ligands indicated at the bottom of the graph. Experimental conditions were identical with Fig. 3A. *p<0.01 vs. IL-6 production in MyD88+/− cells in each corresponding protein concentration. (D) LDH activity was measured in the same culture supernatant that was used for IL-6 ELISA. % cytotoxicity was calculated as a relative LDH activity of maximally released LDH by a treatment of 1% triton X-100. *p<0.01 vs. the other treatments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2722020&req=5

ppat-1000556-g003: PSD-induced proinflammatory response is MyD88-dependent and PSD, in higher concentration, exerts a cytotoxic effect on peritoneal mouse macrophages.(A) IL-6 production in peritoneal macrophages isolated from MyD88+/− (left) or MyD88−/− (right) mouse in response to three purified V. cholerae proteins (PSD, FlaD and VC0222). Cells were treated with the protein in three different final concentrations, 15 µg/ml (black bars), 1.5 µg/ml (gray bars) and 150 ng/ml (hatched bars). Isolation and stimulation of resident murine macrophages were performed as described in Materials and Methods. *p<0.01 vs. IL-6 production in MyD88+/− cells in each corresponding protein concentration. (B) Purity of the protein stimulants was shown in SDS-PAGE. One µg of each protein was separated in 4–12% SDS-PAGE. (C) IL-6 production in peritoneal macrophages isolated from MyD88+/− (left) or MyD88−/− (right) mouse in response to non-protein ligands indicated at the bottom of the graph. Experimental conditions were identical with Fig. 3A. *p<0.01 vs. IL-6 production in MyD88+/− cells in each corresponding protein concentration. (D) LDH activity was measured in the same culture supernatant that was used for IL-6 ELISA. % cytotoxicity was calculated as a relative LDH activity of maximally released LDH by a treatment of 1% triton X-100. *p<0.01 vs. the other treatments.
Mentions: To gain an insight into the signaling mechanism(s) by which PSD activates mouse macrophages, PSD was used to stimulate the induction of the proinflammatory cytokine, IL-6, in MyD88−/− (Myeloid Differentiation Factor-88) and MyD88+/− macrophages. MyD88 is one of most commonly used adaptor molecules that mediates signal transduction in mammalian innate immune activation [30],[31]. Upon ligand binding, TLRs recruit many downstream signaling molecules via MyD88 to activate NF-κB, which then transcribes genes involved in the production of proinflammatory cytokines [1]. LPS is a known potent stimulator of MyD88-dependent inflammatory pathway and stimulates a high level of IL-6 secretion from MyD88 positive macrophages compared to MyD88 negative macrophages [32]. When treated with PSD or LPS, MyD88 knockout macrophage cells secreted significantly less IL-6 compared to the level of IL-6 detected in MyD88 positive macrophages (Fig. 3A and C) indicating that PSD triggers through a predominantly MyD88-dependent inflammatory signaling cascade similar to that observed with LPS.

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