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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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Porcine PBMC and human TLR2/6 stimulation of innate immunity activating fractions.Porcine PBMCs were stimulated with an innate immunity activating fraction of S. suis strain 8067 (concentrated supernatant of penicillin treated bacteria) subdivided into 24 fractions of different molecular sizes. At 4 h post stimulation IL-1β (A) and IL-8 (B) mRNA expression levels were determined by quantitative real time PCR. HeLa 57A cells expressing human TLR2/6 (C), and control cells transfected with vector without insert (D) were stimulated (5 h) with the same 24 fractions. Data represent relative fold activation calculated by dividing the normalized test samples by the normalized activity of medium-stimulated negative control samples. Values represent the mean ± SD of two experiments performed in duplicate. Fractions that induced >5 fold porcine PBMC activation were analyzed by mass spectrometry (Table 1).
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pone-0022299-g002: Porcine PBMC and human TLR2/6 stimulation of innate immunity activating fractions.Porcine PBMCs were stimulated with an innate immunity activating fraction of S. suis strain 8067 (concentrated supernatant of penicillin treated bacteria) subdivided into 24 fractions of different molecular sizes. At 4 h post stimulation IL-1β (A) and IL-8 (B) mRNA expression levels were determined by quantitative real time PCR. HeLa 57A cells expressing human TLR2/6 (C), and control cells transfected with vector without insert (D) were stimulated (5 h) with the same 24 fractions. Data represent relative fold activation calculated by dividing the normalized test samples by the normalized activity of medium-stimulated negative control samples. Values represent the mean ± SD of two experiments performed in duplicate. Fractions that induced >5 fold porcine PBMC activation were analyzed by mass spectrometry (Table 1).

Mentions: To gain more insights into the nature of the porcine PBMC activating component(s), we concentrated the supernatant of penicillin-treated S. suis and size fractionated it into 24 fractions by SDS-PAGE. The obtained fractions were analyzed for their ability to stimulate porcine PBMCs. More than half of the fractions increased IL-1β and IL-8 cytokine transcript levels as measured by qRT-PCR (Fig. 2A, B). The kinetics of the changes in IL-1β and IL-8 mRNA were very similar. The fractions that caused a more than 5-fold increase in IL-1β and IL-8 mRNA were individually analyzed by mass spectrometry. Mascot scores were determined using the identified peptides in all the fractions simultaneously to increase the sensitivity and specificity of the analysis. A total of 24 S. suis proteins with MASCOT scores >50 (Table 1) were identified. Among these 24 proteins, nine (37.5%) putative lipoproteins were present, including two lipoproteins previously shown to be recognized by porcine convalescent sera [19], [20]. In the genome of S. suis strain P1/7, 45 putative lipoprotein coding genes are present (Table S1, [21]) which corresponds to 2.5% of the proteome [21]. This large enrichment of lipoproteins in the porcine PBMC activating fractions suggests that S. suis lipoproteins contribute to the observed PBMC activation.


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 and human TLR2/6 stimulation of innate immunity activating fractions.Porcine PBMCs were stimulated with an innate immunity activating fraction of S. suis strain 8067 (concentrated supernatant of penicillin treated bacteria) subdivided into 24 fractions of different molecular sizes. At 4 h post stimulation IL-1β (A) and IL-8 (B) mRNA expression levels were determined by quantitative real time PCR. HeLa 57A cells expressing human TLR2/6 (C), and control cells transfected with vector without insert (D) were stimulated (5 h) with the same 24 fractions. Data represent relative fold activation calculated by dividing the normalized test samples by the normalized activity of medium-stimulated negative control samples. Values represent the mean ± SD of two experiments performed in duplicate. Fractions that induced >5 fold porcine PBMC activation were analyzed by mass spectrometry (Table 1).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022299-g002: Porcine PBMC and human TLR2/6 stimulation of innate immunity activating fractions.Porcine PBMCs were stimulated with an innate immunity activating fraction of S. suis strain 8067 (concentrated supernatant of penicillin treated bacteria) subdivided into 24 fractions of different molecular sizes. At 4 h post stimulation IL-1β (A) and IL-8 (B) mRNA expression levels were determined by quantitative real time PCR. HeLa 57A cells expressing human TLR2/6 (C), and control cells transfected with vector without insert (D) were stimulated (5 h) with the same 24 fractions. Data represent relative fold activation calculated by dividing the normalized test samples by the normalized activity of medium-stimulated negative control samples. Values represent the mean ± SD of two experiments performed in duplicate. Fractions that induced >5 fold porcine PBMC activation were analyzed by mass spectrometry (Table 1).
Mentions: To gain more insights into the nature of the porcine PBMC activating component(s), we concentrated the supernatant of penicillin-treated S. suis and size fractionated it into 24 fractions by SDS-PAGE. The obtained fractions were analyzed for their ability to stimulate porcine PBMCs. More than half of the fractions increased IL-1β and IL-8 cytokine transcript levels as measured by qRT-PCR (Fig. 2A, B). The kinetics of the changes in IL-1β and IL-8 mRNA were very similar. The fractions that caused a more than 5-fold increase in IL-1β and IL-8 mRNA were individually analyzed by mass spectrometry. Mascot scores were determined using the identified peptides in all the fractions simultaneously to increase the sensitivity and specificity of the analysis. A total of 24 S. suis proteins with MASCOT scores >50 (Table 1) were identified. Among these 24 proteins, nine (37.5%) putative lipoproteins were present, including two lipoproteins previously shown to be recognized by porcine convalescent sera [19], [20]. In the genome of S. suis strain P1/7, 45 putative lipoprotein coding genes are present (Table S1, [21]) which corresponds to 2.5% of the proteome [21]. This large enrichment of lipoproteins in the porcine PBMC activating fractions suggests that S. suis lipoproteins contribute to the observed PBMC activation.

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