Limits...
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.

Show MeSH

Related in: MedlinePlus

Alignment of Orf2 sequences of differentS. suisisolates as available in the database. Phylogenetic tree of ORF2 sequences available in the database. Three different clusters are indicated in dotted boxes, sub-clusters are also indicated. Virulence of strains is indicated using V, AV, WV (virulent, weakly virulent, avirulent), as well as the host where virulence was determined indicatd with p or h (pigs, humans).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4232619&req=5

Fig3: Alignment of Orf2 sequences of differentS. suisisolates as available in the database. Phylogenetic tree of ORF2 sequences available in the database. Three different clusters are indicated in dotted boxes, sub-clusters are also indicated. Virulence of strains is indicated using V, AV, WV (virulent, weakly virulent, avirulent), as well as the host where virulence was determined indicatd with p or h (pigs, humans).

Mentions: Besides the correlation between promoter strength and virulence, we also looked for an association between amino acid sequence of ORF2 and virulence. Sequence analysis of the orf2-folC-operon of wild type strains 10 and S735 revealed several SNPs throughout the sequence [11]. A comparison of ORF2 protein sequences in different isolates revealed more heterogeneity. However, ORF2 protein sequence of all serotype 2 MRP+EF+ isolates were identical to the sequence of ORF2 in strain 10, besides some variation in the predicted start site of the proteins. Clustering of protein sequences revealed three groups of ORF2 sequences, indicated with cluster 1, 2 and 3 in Figure 3. Within cluster 1, two subclusters could be identified: cluster 1A and 1B. Cluster 1A contained strains with an ORF2 sequence that was identical to strain 10; these isolates seem to be associated with virulence. All of the characterized isolates within cluster 1A belong to CC1 and express EF protein. Furthermore, all isolates containing the stronger promoter belonged to this group. Cluster 1B contained two isolates, a Chinese serotype 7 isolate and a serotype 2 MRP−EF− isolate (89–1591), with respectively one and two amino acid substitutions in ORF2 compared to ORF2 of strain 10. The observation that 89–1591 clusters with a serotype 7 isolate, instead of with the other serotype 2 MRP−EF− isolates, adds to the speculation that strain 89–1591 is more similar to serotype 7 isolates than to other MRP−EF− isolates as was also suggested in our CGH study [14]. Within cluster 2, three subclusters could be identified: cluster 2B contained avirulent serotype 2 isolates that were MRP−EF−, cluster 2C contained weakly virulent MRP+EF− isolates including S735, and cluster 2A contained 1 serotype 3 isolate with unknown virulence. Within these subclusters amino acid sequences are identical, the subclusters differ at 13 (2A), 14 (2B) and 9 (2C) amino acid positions compared to strain 10. Cluster 3 contained serotype 9 isolates from different geographical locations, including the reference strain 5218. In conclusion, isolates that are associated with virulence have a strong promoter and an identical protein sequence of ORF2, whereas less virulent isolates have a weaker promoter and a different protein sequence. The relevance of the heterogenic amino acid composition of ORF2 is unknown, it could interfere with function or folding of ORF2.Figure 3


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)

Alignment of Orf2 sequences of differentS. suisisolates as available in the database. Phylogenetic tree of ORF2 sequences available in the database. Three different clusters are indicated in dotted boxes, sub-clusters are also indicated. Virulence of strains is indicated using V, AV, WV (virulent, weakly virulent, avirulent), as well as the host where virulence was determined indicatd with p or h (pigs, humans).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4232619&req=5

Fig3: Alignment of Orf2 sequences of differentS. suisisolates as available in the database. Phylogenetic tree of ORF2 sequences available in the database. Three different clusters are indicated in dotted boxes, sub-clusters are also indicated. Virulence of strains is indicated using V, AV, WV (virulent, weakly virulent, avirulent), as well as the host where virulence was determined indicatd with p or h (pigs, humans).
Mentions: Besides the correlation between promoter strength and virulence, we also looked for an association between amino acid sequence of ORF2 and virulence. Sequence analysis of the orf2-folC-operon of wild type strains 10 and S735 revealed several SNPs throughout the sequence [11]. A comparison of ORF2 protein sequences in different isolates revealed more heterogeneity. However, ORF2 protein sequence of all serotype 2 MRP+EF+ isolates were identical to the sequence of ORF2 in strain 10, besides some variation in the predicted start site of the proteins. Clustering of protein sequences revealed three groups of ORF2 sequences, indicated with cluster 1, 2 and 3 in Figure 3. Within cluster 1, two subclusters could be identified: cluster 1A and 1B. Cluster 1A contained strains with an ORF2 sequence that was identical to strain 10; these isolates seem to be associated with virulence. All of the characterized isolates within cluster 1A belong to CC1 and express EF protein. Furthermore, all isolates containing the stronger promoter belonged to this group. Cluster 1B contained two isolates, a Chinese serotype 7 isolate and a serotype 2 MRP−EF− isolate (89–1591), with respectively one and two amino acid substitutions in ORF2 compared to ORF2 of strain 10. The observation that 89–1591 clusters with a serotype 7 isolate, instead of with the other serotype 2 MRP−EF− isolates, adds to the speculation that strain 89–1591 is more similar to serotype 7 isolates than to other MRP−EF− isolates as was also suggested in our CGH study [14]. Within cluster 2, three subclusters could be identified: cluster 2B contained avirulent serotype 2 isolates that were MRP−EF−, cluster 2C contained weakly virulent MRP+EF− isolates including S735, and cluster 2A contained 1 serotype 3 isolate with unknown virulence. Within these subclusters amino acid sequences are identical, the subclusters differ at 13 (2A), 14 (2B) and 9 (2C) amino acid positions compared to strain 10. Cluster 3 contained serotype 9 isolates from different geographical locations, including the reference strain 5218. In conclusion, isolates that are associated with virulence have a strong promoter and an identical protein sequence of ORF2, whereas less virulent isolates have a weaker promoter and a different protein sequence. The relevance of the heterogenic amino acid composition of ORF2 is unknown, it could interfere with function or folding of ORF2.Figure 3

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