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A naturally occurring nucleotide polymorphism in the orf2/folc promoter is associated with Streptococcus suis virulence.

de Greeff A, Buys H, Wells JM, Smith HE - BMC Microbiol. (2014)

Bottom Line: This increase in virulence could not be associated with changes in pro-inflammatory responses of porcine blood mononucleated cells in response to S. suis in vitro.Sequence analysis of the orf2-folC-operon of S. suis isolates 10 and S735 revealed an SNP in the -35 region of the putative promoter sequence of the operon, as well as several SNPs resulting in amino acid substitutions in the ORF2 protein.In summary, we demonstrate the importance of orf2 in the virulence of S. suis.

View Article: PubMed Central - PubMed

Affiliation: Central Veterinary Institute of Wageningen UR, Edelhertweg 15, 8219, , PH, Lelystad, The Netherlands. astrid.degreeff@wur.nl.

ABSTRACT

Background: Streptococcus suis is a major problem in the swine industry causing meningitis, arthritis and pericarditis in piglets. Pathogenesis of S. suis is poorly understood. We previously showed that introduction of a 3 kb genomic fragment from virulent serotype 2 strain 10 into a weakly virulent serotype 2 strain S735, generated a hypervirulent isolate. The 3 kb genomic fragment contained two complete open reading frames (ORF) in an operon-structure of which one ORF showed similarity to folylpolyglutamate synthetase, whereas the function of the second ORF could not be predicted based on database searches for protein similarity.

Results: In this study we demonstrate that introduction of orf2 from strain 10 into strain S735 is sufficient to dramatically increase the virulence of S735 in pigs. This increase in virulence could not be associated with changes in pro-inflammatory responses of porcine blood mononucleated cells in response to S. suis in vitro. Sequence analysis of the orf2-folC-operon of S. suis isolates 10 and S735 revealed an SNP in the -35 region of the putative promoter sequence of the operon, as well as several SNPs resulting in amino acid substitutions in the ORF2 protein. Transcript levels of orf2 and folC were significantly higher in the virulent strain 10 than in the weakly virulent strain S735 and in vitro mutagenesis of the orf2 promoter confirmed that this was due to a SNP in the predicted -35 region upstream of the orf2 promoter. In this study, we demonstrated that the stronger promoter was present in all virulent and highly virulent S. suis isolates included in our study. This highlights a correlation between high orf2 expression and virulence. Conversely, the weaker promoter was present in isolates known to be weakly pathogenic or non-pathogenic.

Conclusion: In summary, we demonstrate the importance of orf2 in the virulence of S. suis.

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Related in: MedlinePlus

Innate immune response of porcine PBMCs toS. suisisolates. Porcine PBMCs were incubated with S. suis strains S735-pCOM1 and S735-pCOM1-orf2[10] at an MOI of 1. Gene expression of IL-1-β (panel A), IL-6 (panel B), IL-8 (panel C), IL-10 (panel D), TNF-α (panel E), IFN-γ (panel F), and IL-12 (panel G) was determined using qPCR after 2 h (white bars), 4 h (hatched bars) and 6 h (black bars) of stimulation. Relative expression was determined by expressing the amount of target gene relative to a housekeeping gene. LPS: lipopolysaccharide; PBS: phosphate buffered saline. Each bar represents two individual experiments each performed in duplo. Error bars represent standard error of the mean. Significance was determined by 2-way ANOVA analysis, only significant differences between PBS treatment, S735-pCOM1 and S735-pCOM1-orf2[10] are indicated, *p <0.05; **p <0.01; ***p <0.001.
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Fig1: Innate immune response of porcine PBMCs toS. suisisolates. Porcine PBMCs were incubated with S. suis strains S735-pCOM1 and S735-pCOM1-orf2[10] at an MOI of 1. Gene expression of IL-1-β (panel A), IL-6 (panel B), IL-8 (panel C), IL-10 (panel D), TNF-α (panel E), IFN-γ (panel F), and IL-12 (panel G) was determined using qPCR after 2 h (white bars), 4 h (hatched bars) and 6 h (black bars) of stimulation. Relative expression was determined by expressing the amount of target gene relative to a housekeeping gene. LPS: lipopolysaccharide; PBS: phosphate buffered saline. Each bar represents two individual experiments each performed in duplo. Error bars represent standard error of the mean. Significance was determined by 2-way ANOVA analysis, only significant differences between PBS treatment, S735-pCOM1 and S735-pCOM1-orf2[10] are indicated, *p <0.05; **p <0.01; ***p <0.001.

Mentions: As clinical signs of S. suis strains S735-pCOM1-V[10] and S735-pCOM1-orf2[10] were severe, it was hypothesized that introduction of orf2 might exacerbate the innate inflammatory response to S. suis contributing to the host pathology and symptoms. To test this hypothesis, immune responses of porcine PBMCs to S735-pCOM1-orf2[10] and S735-pCOM1 were compared in vitro. Gene expression levels of innate immune genes of PBMCs were determined after incubation with S. suis isolates as a function of time (2, 4 and 6 h p.i.) using qPCR. Both S735-pCOM1 and S735-pCOM1-orf2[10] induced high gene expression of pro-inflammatory cytokine IL-1-β (170-fold) and chemokine IL-8 (130-fold) compared to controls whereas relatively low gene expression of pro-inflammatory IL-6 (15-fold), anti-inflammatory IL-10 (8-fold), and TNF-α (9-fold) and IFN-γ (4-fold) was induced by both isolates (Figure 1). The transcript levels peaked at 4 h p.i. for all genes tested except for IL-12 which was not expressed in response to S. suis during the incubation time of our experiment (Figure 1). There were no significant differences between the two isolates in the expression levels of any of the tested immune genes. These data suggest that the observed increased virulence of S735-pCOM1-orf2[10] in piglets compared to S735-pCOM1 is probably not due to differences in the host innate responses to this strain.Figure 1


A naturally occurring nucleotide polymorphism in the orf2/folc promoter is associated with Streptococcus suis virulence.

de Greeff A, Buys H, Wells JM, Smith HE - BMC Microbiol. (2014)

Innate immune response of porcine PBMCs toS. suisisolates. Porcine PBMCs were incubated with S. suis strains S735-pCOM1 and S735-pCOM1-orf2[10] at an MOI of 1. Gene expression of IL-1-β (panel A), IL-6 (panel B), IL-8 (panel C), IL-10 (panel D), TNF-α (panel E), IFN-γ (panel F), and IL-12 (panel G) was determined using qPCR after 2 h (white bars), 4 h (hatched bars) and 6 h (black bars) of stimulation. Relative expression was determined by expressing the amount of target gene relative to a housekeeping gene. LPS: lipopolysaccharide; PBS: phosphate buffered saline. Each bar represents two individual experiments each performed in duplo. Error bars represent standard error of the mean. Significance was determined by 2-way ANOVA analysis, only significant differences between PBS treatment, S735-pCOM1 and S735-pCOM1-orf2[10] are indicated, *p <0.05; **p <0.01; ***p <0.001.
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Related In: Results  -  Collection

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Fig1: Innate immune response of porcine PBMCs toS. suisisolates. Porcine PBMCs were incubated with S. suis strains S735-pCOM1 and S735-pCOM1-orf2[10] at an MOI of 1. Gene expression of IL-1-β (panel A), IL-6 (panel B), IL-8 (panel C), IL-10 (panel D), TNF-α (panel E), IFN-γ (panel F), and IL-12 (panel G) was determined using qPCR after 2 h (white bars), 4 h (hatched bars) and 6 h (black bars) of stimulation. Relative expression was determined by expressing the amount of target gene relative to a housekeeping gene. LPS: lipopolysaccharide; PBS: phosphate buffered saline. Each bar represents two individual experiments each performed in duplo. Error bars represent standard error of the mean. Significance was determined by 2-way ANOVA analysis, only significant differences between PBS treatment, S735-pCOM1 and S735-pCOM1-orf2[10] are indicated, *p <0.05; **p <0.01; ***p <0.001.
Mentions: As clinical signs of S. suis strains S735-pCOM1-V[10] and S735-pCOM1-orf2[10] were severe, it was hypothesized that introduction of orf2 might exacerbate the innate inflammatory response to S. suis contributing to the host pathology and symptoms. To test this hypothesis, immune responses of porcine PBMCs to S735-pCOM1-orf2[10] and S735-pCOM1 were compared in vitro. Gene expression levels of innate immune genes of PBMCs were determined after incubation with S. suis isolates as a function of time (2, 4 and 6 h p.i.) using qPCR. Both S735-pCOM1 and S735-pCOM1-orf2[10] induced high gene expression of pro-inflammatory cytokine IL-1-β (170-fold) and chemokine IL-8 (130-fold) compared to controls whereas relatively low gene expression of pro-inflammatory IL-6 (15-fold), anti-inflammatory IL-10 (8-fold), and TNF-α (9-fold) and IFN-γ (4-fold) was induced by both isolates (Figure 1). The transcript levels peaked at 4 h p.i. for all genes tested except for IL-12 which was not expressed in response to S. suis during the incubation time of our experiment (Figure 1). There were no significant differences between the two isolates in the expression levels of any of the tested immune genes. These data suggest that the observed increased virulence of S735-pCOM1-orf2[10] in piglets compared to S735-pCOM1 is probably not due to differences in the host innate responses to this strain.Figure 1

Bottom Line: This increase in virulence could not be associated with changes in pro-inflammatory responses of porcine blood mononucleated cells in response to S. suis in vitro.Sequence analysis of the orf2-folC-operon of S. suis isolates 10 and S735 revealed an SNP in the -35 region of the putative promoter sequence of the operon, as well as several SNPs resulting in amino acid substitutions in the ORF2 protein.In summary, we demonstrate the importance of orf2 in the virulence of S. suis.

View Article: PubMed Central - PubMed

Affiliation: Central Veterinary Institute of Wageningen UR, Edelhertweg 15, 8219, , PH, Lelystad, The Netherlands. astrid.degreeff@wur.nl.

ABSTRACT

Background: Streptococcus suis is a major problem in the swine industry causing meningitis, arthritis and pericarditis in piglets. Pathogenesis of S. suis is poorly understood. We previously showed that introduction of a 3 kb genomic fragment from virulent serotype 2 strain 10 into a weakly virulent serotype 2 strain S735, generated a hypervirulent isolate. The 3 kb genomic fragment contained two complete open reading frames (ORF) in an operon-structure of which one ORF showed similarity to folylpolyglutamate synthetase, whereas the function of the second ORF could not be predicted based on database searches for protein similarity.

Results: In this study we demonstrate that introduction of orf2 from strain 10 into strain S735 is sufficient to dramatically increase the virulence of S735 in pigs. This increase in virulence could not be associated with changes in pro-inflammatory responses of porcine blood mononucleated cells in response to S. suis in vitro. Sequence analysis of the orf2-folC-operon of S. suis isolates 10 and S735 revealed an SNP in the -35 region of the putative promoter sequence of the operon, as well as several SNPs resulting in amino acid substitutions in the ORF2 protein. Transcript levels of orf2 and folC were significantly higher in the virulent strain 10 than in the weakly virulent strain S735 and in vitro mutagenesis of the orf2 promoter confirmed that this was due to a SNP in the predicted -35 region upstream of the orf2 promoter. In this study, we demonstrated that the stronger promoter was present in all virulent and highly virulent S. suis isolates included in our study. This highlights a correlation between high orf2 expression and virulence. Conversely, the weaker promoter was present in isolates known to be weakly pathogenic or non-pathogenic.

Conclusion: In summary, we demonstrate the importance of orf2 in the virulence of S. suis.

Show MeSH
Related in: MedlinePlus