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Lgt processing is an essential step in Streptococcus suis lipoprotein mediated innate immune activation.

Wichgers Schreur PJ, Rebel JM, Smits MA, van Putten JP, Smith HE - PLoS ONE (2011)

Bottom Line: Genetic inactivation of a putative prolipoprotein diacylglyceryl transferase (Lgt) gene resulted in deficient lipoprotein synthesis as evidenced by palmitate labeling.The Lgt mutant showed strongly reduced activation of porcine PBMCs, indicating that lipoproteins are dominant porcine PBMC activating molecules of S. suis.In addition, we provide evidence that Lgt processing of lipoproteins is required for lipoprotein mediated innate immune activation.

View Article: PubMed Central - PubMed

Affiliation: Central Veterinary Institute, Wageningen UR, Lelystad, The Netherlands. paul.wichgersschreur@wur.nl

ABSTRACT

Background: Streptococcus suis causes invasive infections in pigs and occasionally in humans. The host innate immune system plays a major role in counteracting S. suis infections. The main components of S. suis able to activate the innate immune system likely include cell wall constituents that may be released during growth or after cell wall integrity loss, however characterization of these components is still limited.

Methodology/principal findings: [corrected] A concentrated very potent innate immunity activating supernatant of penicillin-treated S. suis was SDS-PAGE fractionated and tested for porcine peripheral blood mononucleated cell (PBMC) stimulating activity using cytokine gene transcript analysis. More than half of the 24 tested fractions increased IL-1β and IL-8 cytokine gene transcript levels in porcine PBMCs. Mass spectrometry of the active fractions indicated 24 proteins including 9 lipoproteins. Genetic inactivation of a putative prolipoprotein diacylglyceryl transferase (Lgt) gene resulted in deficient lipoprotein synthesis as evidenced by palmitate labeling. The Lgt mutant showed strongly reduced activation of porcine PBMCs, indicating that lipoproteins are dominant porcine PBMC activating molecules of S. suis.

Conclusion/significance: This study for the first time identifies and characterizes lipoproteins of S. suis as major activators of the innate immune system of the pig. In addition, we provide evidence that Lgt processing of lipoproteins is required for lipoprotein mediated innate immune activation.

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Related in: MedlinePlus

Porcine PBMC activating capacity of wild type and Δlgt mutant bacteria.Porcine PBMCs were stimulated with wild type, Δlgt, Δlgt::pGA14-cm, and Δlgt::pGA14-lgt mutant bacteria. PBMCs were stimulated with supernatants derived from penicillin treated bacteria (A, B), cells in the presence of penicillin (C, D), supernatants of stationary phase grown bacteria in the absence of penicillin (E, F) and cells in the absence of penicillin (G, H). At 2 h (light grey bar) and 4 h (dark grey bar) after stimulation, IL-1β (A, C, E, G) and IL-8 (B, D, F, H) mRNA levels were determined. The diacylated lipopeptide FSL was used as a positive control. Data represent relative fold activation calculated by dividing the normalized activity of the test samples by the normalized activity of medium-stimulated negative control samples. Values represent the mean ± SD of three independent experiments performed in duplicate. * P<0.05 compared to wild type level.
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pone-0022299-g006: Porcine PBMC activating capacity of wild type and Δlgt mutant bacteria.Porcine PBMCs were stimulated with wild type, Δlgt, Δlgt::pGA14-cm, and Δlgt::pGA14-lgt mutant bacteria. PBMCs were stimulated with supernatants derived from penicillin treated bacteria (A, B), cells in the presence of penicillin (C, D), supernatants of stationary phase grown bacteria in the absence of penicillin (E, F) and cells in the absence of penicillin (G, H). At 2 h (light grey bar) and 4 h (dark grey bar) after stimulation, IL-1β (A, C, E, G) and IL-8 (B, D, F, H) mRNA levels were determined. The diacylated lipopeptide FSL was used as a positive control. Data represent relative fold activation calculated by dividing the normalized activity of the test samples by the normalized activity of medium-stimulated negative control samples. Values represent the mean ± SD of three independent experiments performed in duplicate. * P<0.05 compared to wild type level.

Mentions: In contrast to the transfected HeLa cells expressing human TLR2/6, porcine PBMC express multiple innate immune receptors that may respond to various S. suis components. To assess the contribution of lipoproteins to PBMC activation, we stimulated porcine PBMCs with (penicillin-treated) supernatants and cells of wild type, Δlgt mutant, Δlgt::pGA14-cm mutant and Δlgt::pGA14-lgt mutant bacteria. Stimulation with the wild type and the Δlgt::pGA14-lgt mutant bacterial supernatants resulted in efficient induction of IL-1β and IL-8 mRNA at 2 h and 4 h post stimulation (Figs. 6A and B). As expected, only minimal induction of IL-1β and IL-8 mRNA was observed after stimulation with the Δlgt mutant and Δlgt::pGA14-cm mutant derived supernatant. In line with the activation kinetics of the supernatants, PBMCs stimulation with wild type and Δlgt::pGA14-lgt mutant bacteria also resulted in efficient induction of IL-1β and IL-8 mRNA at 2 h and 4 h post stimulation (Fig. 6C and D). The IL-1β and IL-8 mRNA levels induced by the Δlgt mutant and the Δlgt::pGA14-cm mutant were once more strongly reduced compared to the wild type strain especially at 2 h post stimulation. These results suggest S. suis lipoproteins as the principal activators of the porcine PBMC innate immune response.


Lgt processing is an essential step in Streptococcus suis lipoprotein mediated innate immune activation.

Wichgers Schreur PJ, Rebel JM, Smits MA, van Putten JP, Smith HE - PLoS ONE (2011)

Porcine PBMC activating capacity of wild type and Δlgt mutant bacteria.Porcine PBMCs were stimulated with wild type, Δlgt, Δlgt::pGA14-cm, and Δlgt::pGA14-lgt mutant bacteria. PBMCs were stimulated with supernatants derived from penicillin treated bacteria (A, B), cells in the presence of penicillin (C, D), supernatants of stationary phase grown bacteria in the absence of penicillin (E, F) and cells in the absence of penicillin (G, H). At 2 h (light grey bar) and 4 h (dark grey bar) after stimulation, IL-1β (A, C, E, G) and IL-8 (B, D, F, H) mRNA levels were determined. The diacylated lipopeptide FSL was used as a positive control. Data represent relative fold activation calculated by dividing the normalized activity of the test samples by the normalized activity of medium-stimulated negative control samples. Values represent the mean ± SD of three independent experiments performed in duplicate. * P<0.05 compared to wild type level.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139625&req=5

pone-0022299-g006: Porcine PBMC activating capacity of wild type and Δlgt mutant bacteria.Porcine PBMCs were stimulated with wild type, Δlgt, Δlgt::pGA14-cm, and Δlgt::pGA14-lgt mutant bacteria. PBMCs were stimulated with supernatants derived from penicillin treated bacteria (A, B), cells in the presence of penicillin (C, D), supernatants of stationary phase grown bacteria in the absence of penicillin (E, F) and cells in the absence of penicillin (G, H). At 2 h (light grey bar) and 4 h (dark grey bar) after stimulation, IL-1β (A, C, E, G) and IL-8 (B, D, F, H) mRNA levels were determined. The diacylated lipopeptide FSL was used as a positive control. Data represent relative fold activation calculated by dividing the normalized activity of the test samples by the normalized activity of medium-stimulated negative control samples. Values represent the mean ± SD of three independent experiments performed in duplicate. * P<0.05 compared to wild type level.
Mentions: In contrast to the transfected HeLa cells expressing human TLR2/6, porcine PBMC express multiple innate immune receptors that may respond to various S. suis components. To assess the contribution of lipoproteins to PBMC activation, we stimulated porcine PBMCs with (penicillin-treated) supernatants and cells of wild type, Δlgt mutant, Δlgt::pGA14-cm mutant and Δlgt::pGA14-lgt mutant bacteria. Stimulation with the wild type and the Δlgt::pGA14-lgt mutant bacterial supernatants resulted in efficient induction of IL-1β and IL-8 mRNA at 2 h and 4 h post stimulation (Figs. 6A and B). As expected, only minimal induction of IL-1β and IL-8 mRNA was observed after stimulation with the Δlgt mutant and Δlgt::pGA14-cm mutant derived supernatant. In line with the activation kinetics of the supernatants, PBMCs stimulation with wild type and Δlgt::pGA14-lgt mutant bacteria also resulted in efficient induction of IL-1β and IL-8 mRNA at 2 h and 4 h post stimulation (Fig. 6C and D). The IL-1β and IL-8 mRNA levels induced by the Δlgt mutant and the Δlgt::pGA14-cm mutant were once more strongly reduced compared to the wild type strain especially at 2 h post stimulation. These results suggest S. suis lipoproteins as the principal activators of the porcine PBMC innate immune response.

Bottom Line: Genetic inactivation of a putative prolipoprotein diacylglyceryl transferase (Lgt) gene resulted in deficient lipoprotein synthesis as evidenced by palmitate labeling.The Lgt mutant showed strongly reduced activation of porcine PBMCs, indicating that lipoproteins are dominant porcine PBMC activating molecules of S. suis.In addition, we provide evidence that Lgt processing of lipoproteins is required for lipoprotein mediated innate immune activation.

View Article: PubMed Central - PubMed

Affiliation: Central Veterinary Institute, Wageningen UR, Lelystad, The Netherlands. paul.wichgersschreur@wur.nl

ABSTRACT

Background: Streptococcus suis causes invasive infections in pigs and occasionally in humans. The host innate immune system plays a major role in counteracting S. suis infections. The main components of S. suis able to activate the innate immune system likely include cell wall constituents that may be released during growth or after cell wall integrity loss, however characterization of these components is still limited.

Methodology/principal findings: [corrected] A concentrated very potent innate immunity activating supernatant of penicillin-treated S. suis was SDS-PAGE fractionated and tested for porcine peripheral blood mononucleated cell (PBMC) stimulating activity using cytokine gene transcript analysis. More than half of the 24 tested fractions increased IL-1β and IL-8 cytokine gene transcript levels in porcine PBMCs. Mass spectrometry of the active fractions indicated 24 proteins including 9 lipoproteins. Genetic inactivation of a putative prolipoprotein diacylglyceryl transferase (Lgt) gene resulted in deficient lipoprotein synthesis as evidenced by palmitate labeling. The Lgt mutant showed strongly reduced activation of porcine PBMCs, indicating that lipoproteins are dominant porcine PBMC activating molecules of S. suis.

Conclusion/significance: This study for the first time identifies and characterizes lipoproteins of S. suis as major activators of the innate immune system of the pig. In addition, we provide evidence that Lgt processing of lipoproteins is required for lipoprotein mediated innate immune activation.

Show MeSH
Related in: MedlinePlus