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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

Growth of wild type and Δlgt mutant bacteria.Growth of wild type, Δlgt mutant, and the complemented Δlgt mutant (Δlgt::pGA14-cm; Δlgt::pGA14-lgt) bacteria was assessed in THB by following optical densities in time.
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pone-0022299-g003: Growth of wild type and Δlgt mutant bacteria.Growth of wild type, Δlgt mutant, and the complemented Δlgt mutant (Δlgt::pGA14-cm; Δlgt::pGA14-lgt) bacteria was assessed in THB by following optical densities in time.

Mentions: To distinguish between lipoprotein and non-lipoprotein mediated innate immune activation of porcine PBMCs, we constructed a mutant S. suis serotype 9 isolate deficient in the expression of the lipoprotein processing enzyme Lgt. Lgt in Gram-positive bacteria is required for lipid modification of the cysteine residue present within the lipobox of prelipoproteins. In the genome of S. suis serotype 2 strain P1/7 gene SSU_1418 had been annotated to encode the Lgt protein. This putative Lgt protein showed 67% amino acid sequence identity to the Lgt protein of S. pneumoniae strain D39 [23]. The lgt gene is the second gene transcribed of an operon expressing 4 genes also encoding two putative exported proteins and a phosphorylase enzyme. We inactivated the corresponding lgt gene in S. suis serotype 9 strain 8067 by homologous recombination generating Δlgt mutant bacteria. A positive control was made by re-introducing an intact lgt gene in the Δlgt mutant strain by plasmid complementation generating Δlgt::pGA14-lgt. As a negative control we complemented the Δlgt mutant with vector lacking the lgt insert, generating Δlgt::pGA14-cm. Inactivation of lgt resulted in viable S. suis bacteria able to grow efficiently in THB after a slightly increased lag phase (Fig. 3).


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)

Growth of wild type and Δlgt mutant bacteria.Growth of wild type, Δlgt mutant, and the complemented Δlgt mutant (Δlgt::pGA14-cm; Δlgt::pGA14-lgt) bacteria was assessed in THB by following optical densities in time.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022299-g003: Growth of wild type and Δlgt mutant bacteria.Growth of wild type, Δlgt mutant, and the complemented Δlgt mutant (Δlgt::pGA14-cm; Δlgt::pGA14-lgt) bacteria was assessed in THB by following optical densities in time.
Mentions: To distinguish between lipoprotein and non-lipoprotein mediated innate immune activation of porcine PBMCs, we constructed a mutant S. suis serotype 9 isolate deficient in the expression of the lipoprotein processing enzyme Lgt. Lgt in Gram-positive bacteria is required for lipid modification of the cysteine residue present within the lipobox of prelipoproteins. In the genome of S. suis serotype 2 strain P1/7 gene SSU_1418 had been annotated to encode the Lgt protein. This putative Lgt protein showed 67% amino acid sequence identity to the Lgt protein of S. pneumoniae strain D39 [23]. The lgt gene is the second gene transcribed of an operon expressing 4 genes also encoding two putative exported proteins and a phosphorylase enzyme. We inactivated the corresponding lgt gene in S. suis serotype 9 strain 8067 by homologous recombination generating Δlgt mutant bacteria. A positive control was made by re-introducing an intact lgt gene in the Δlgt mutant strain by plasmid complementation generating Δlgt::pGA14-lgt. As a negative control we complemented the Δlgt mutant with vector lacking the lgt insert, generating Δlgt::pGA14-cm. Inactivation of lgt resulted in viable S. suis bacteria able to grow efficiently in THB after a slightly increased lag phase (Fig. 3).

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