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Serotype- and strain- dependent contribution of the sensor kinase CovS of the CovRS two-component system to Streptococcus pyogenes pathogenesis.

Sugareva V, Arlt R, Fiedler T, Riani C, Podbielski A, Kreikemeyer B - BMC Microbiol. (2010)

Bottom Line: However, a serotype- and even strain-dependent contribution on survival in whole human blood and biofilm formation was noted, respectively.These data provide new information on the action of the CovS sensor kinase and revealed that its activity on capsule expression and keratinocyte adherence is uniform across serotypes, whereas the influence on biofilm formation and blood survival is serotype or even strain dependent.This adds the CovRS system to a growing list of serotype-specific acting regulatory loci in S. pyogenes.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Rostock, Medical Faculty, Institute of Medical Microbiology, Virology and Hygiene, Department of Med, Microbiology and Hospital Hygiene, Schillingallee 70, 18055 Rostock, Germany.

ABSTRACT

Background: The Streptococcus pyogenes (group A streptococci, GAS) two-component signal transduction system CovRS has been described to be important for pathogenesis of this exclusively human bacterial species. If this system acts uniquely in all serotypes is currently unclear. Presence of serotype- or strain-dependent regulatory circuits and polarity is an emerging scheme in Streptococcus pyogenes pathogenesis. Thus, the contribution of the sensor kinase (CovS) of the global regulatory two-component signal transduction system CovRS on pathogenesis of several M serotypes was investigated.

Results: CovS mutation uniformly repressed capsule expression and hampered keratinocyte adherence in all tested serotypes. However, a serotype- and even strain-dependent contribution on survival in whole human blood and biofilm formation was noted, respectively.

Conclusions: These data provide new information on the action of the CovS sensor kinase and revealed that its activity on capsule expression and keratinocyte adherence is uniform across serotypes, whereas the influence on biofilm formation and blood survival is serotype or even strain dependent. This adds the CovRS system to a growing list of serotype-specific acting regulatory loci in S. pyogenes.

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Schematic representation of the inactivation of CovS. A. Inactivation of CovS in the serotype M49 strain 591. Plasmid pUCerm::covS contains a fragment internal of covRS and confers erythromycin resistance (EmR). The genomic regions from both covR and covS used for recombination are marked in black. B. M49 covRS locus after insertion of the plasmid pUCerm::covS. The thin arrows depict primers used for RT-PCR analysis (see below). The thick numbered arrows (1-4) represent primers used for PCR of whole region and junction fragments to confirm plasmid integration into the chromosome. C. RT-PCR analysis. Primer pairs derived from covR and covS were used. Lane DNA Ladder, O'GeneRuler 1 kb DNA Ladder (Fermentas); gDNA, genomic DNA; cDNA, first-strand synthesized cDNA; mRNA, messenger RNA; -C, negative control, where no template for polymerization was used.
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Figure 1: Schematic representation of the inactivation of CovS. A. Inactivation of CovS in the serotype M49 strain 591. Plasmid pUCerm::covS contains a fragment internal of covRS and confers erythromycin resistance (EmR). The genomic regions from both covR and covS used for recombination are marked in black. B. M49 covRS locus after insertion of the plasmid pUCerm::covS. The thin arrows depict primers used for RT-PCR analysis (see below). The thick numbered arrows (1-4) represent primers used for PCR of whole region and junction fragments to confirm plasmid integration into the chromosome. C. RT-PCR analysis. Primer pairs derived from covR and covS were used. Lane DNA Ladder, O'GeneRuler 1 kb DNA Ladder (Fermentas); gDNA, genomic DNA; cDNA, first-strand synthesized cDNA; mRNA, messenger RNA; -C, negative control, where no template for polymerization was used.

Mentions: Using S. pyogenes M49 chromosomal DNA as a template, a 1023 bp internal fragment of covRS, spanning 663 bp from the covR gene and 355 bp from covS gene, was amplified by PCR employing primers Csrko_for_HindIII (5'-GGCGGCAAGCTTGAAGATGAAAAGAATCTGG-3') and Csrko_rev_BamHI (5'-GGCGGCGGATCCAGACATAAATATCTTGATTCG-3'). The internal fragment was digested by HindIII and BamHI and subsequently cloned into pUCerm [24] to obtain a plasmid designated pUCerm::covS. For electroporation, thawed electrocompetent cells (100 μl) were initially mixed on ice with 10 μl pUCerm::covS. The mixture was next transferred to a pre-chilled 2 mm electrode spacing cuvette (Bio-Rad). Electroporation was then performed using a Gene Pulser II electroporator (Bio-Rad) with the following settings: voltage 1750 V, capacitance 25 μF, 12 ms, 481Ω. Subsequently, 1 ml pre-warmed Todd-Hewitt broth (Invitrogen) supplemented with 0.5% yeast extract and 0.125 M sucrose was added to the transformed cells, and the suspension was incubated at 37°C for 2 h. Transformants were selected on THB agar plates supplemented with 0.5% yeast extract and 5 μg/ml erythromycin. Successful integration of the plasmid was confirmed by PCR analysis of junction fragments using standard protocols (for the used primer locations please refer to fig. 1 and the result section). The generated insertional mutant strains were designated M18::covS, M18_588::covS, M49::covS, M49_581::covS, M49_634::covS, M2::covS, M2_583::covS, M6::covS, M6_586::covS, M6_796::covS and M6_576::covS.


Serotype- and strain- dependent contribution of the sensor kinase CovS of the CovRS two-component system to Streptococcus pyogenes pathogenesis.

Sugareva V, Arlt R, Fiedler T, Riani C, Podbielski A, Kreikemeyer B - BMC Microbiol. (2010)

Schematic representation of the inactivation of CovS. A. Inactivation of CovS in the serotype M49 strain 591. Plasmid pUCerm::covS contains a fragment internal of covRS and confers erythromycin resistance (EmR). The genomic regions from both covR and covS used for recombination are marked in black. B. M49 covRS locus after insertion of the plasmid pUCerm::covS. The thin arrows depict primers used for RT-PCR analysis (see below). The thick numbered arrows (1-4) represent primers used for PCR of whole region and junction fragments to confirm plasmid integration into the chromosome. C. RT-PCR analysis. Primer pairs derived from covR and covS were used. Lane DNA Ladder, O'GeneRuler 1 kb DNA Ladder (Fermentas); gDNA, genomic DNA; cDNA, first-strand synthesized cDNA; mRNA, messenger RNA; -C, negative control, where no template for polymerization was used.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2823723&req=5

Figure 1: Schematic representation of the inactivation of CovS. A. Inactivation of CovS in the serotype M49 strain 591. Plasmid pUCerm::covS contains a fragment internal of covRS and confers erythromycin resistance (EmR). The genomic regions from both covR and covS used for recombination are marked in black. B. M49 covRS locus after insertion of the plasmid pUCerm::covS. The thin arrows depict primers used for RT-PCR analysis (see below). The thick numbered arrows (1-4) represent primers used for PCR of whole region and junction fragments to confirm plasmid integration into the chromosome. C. RT-PCR analysis. Primer pairs derived from covR and covS were used. Lane DNA Ladder, O'GeneRuler 1 kb DNA Ladder (Fermentas); gDNA, genomic DNA; cDNA, first-strand synthesized cDNA; mRNA, messenger RNA; -C, negative control, where no template for polymerization was used.
Mentions: Using S. pyogenes M49 chromosomal DNA as a template, a 1023 bp internal fragment of covRS, spanning 663 bp from the covR gene and 355 bp from covS gene, was amplified by PCR employing primers Csrko_for_HindIII (5'-GGCGGCAAGCTTGAAGATGAAAAGAATCTGG-3') and Csrko_rev_BamHI (5'-GGCGGCGGATCCAGACATAAATATCTTGATTCG-3'). The internal fragment was digested by HindIII and BamHI and subsequently cloned into pUCerm [24] to obtain a plasmid designated pUCerm::covS. For electroporation, thawed electrocompetent cells (100 μl) were initially mixed on ice with 10 μl pUCerm::covS. The mixture was next transferred to a pre-chilled 2 mm electrode spacing cuvette (Bio-Rad). Electroporation was then performed using a Gene Pulser II electroporator (Bio-Rad) with the following settings: voltage 1750 V, capacitance 25 μF, 12 ms, 481Ω. Subsequently, 1 ml pre-warmed Todd-Hewitt broth (Invitrogen) supplemented with 0.5% yeast extract and 0.125 M sucrose was added to the transformed cells, and the suspension was incubated at 37°C for 2 h. Transformants were selected on THB agar plates supplemented with 0.5% yeast extract and 5 μg/ml erythromycin. Successful integration of the plasmid was confirmed by PCR analysis of junction fragments using standard protocols (for the used primer locations please refer to fig. 1 and the result section). The generated insertional mutant strains were designated M18::covS, M18_588::covS, M49::covS, M49_581::covS, M49_634::covS, M2::covS, M2_583::covS, M6::covS, M6_586::covS, M6_796::covS and M6_576::covS.

Bottom Line: However, a serotype- and even strain-dependent contribution on survival in whole human blood and biofilm formation was noted, respectively.These data provide new information on the action of the CovS sensor kinase and revealed that its activity on capsule expression and keratinocyte adherence is uniform across serotypes, whereas the influence on biofilm formation and blood survival is serotype or even strain dependent.This adds the CovRS system to a growing list of serotype-specific acting regulatory loci in S. pyogenes.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Rostock, Medical Faculty, Institute of Medical Microbiology, Virology and Hygiene, Department of Med, Microbiology and Hospital Hygiene, Schillingallee 70, 18055 Rostock, Germany.

ABSTRACT

Background: The Streptococcus pyogenes (group A streptococci, GAS) two-component signal transduction system CovRS has been described to be important for pathogenesis of this exclusively human bacterial species. If this system acts uniquely in all serotypes is currently unclear. Presence of serotype- or strain-dependent regulatory circuits and polarity is an emerging scheme in Streptococcus pyogenes pathogenesis. Thus, the contribution of the sensor kinase (CovS) of the global regulatory two-component signal transduction system CovRS on pathogenesis of several M serotypes was investigated.

Results: CovS mutation uniformly repressed capsule expression and hampered keratinocyte adherence in all tested serotypes. However, a serotype- and even strain-dependent contribution on survival in whole human blood and biofilm formation was noted, respectively.

Conclusions: These data provide new information on the action of the CovS sensor kinase and revealed that its activity on capsule expression and keratinocyte adherence is uniform across serotypes, whereas the influence on biofilm formation and blood survival is serotype or even strain dependent. This adds the CovRS system to a growing list of serotype-specific acting regulatory loci in S. pyogenes.

Show MeSH
Related in: MedlinePlus