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SalK/SalR, a two-component signal transduction system, is essential for full virulence of highly invasive Streptococcus suis serotype 2.

Li M, Wang C, Feng Y, Pan X, Cheng G, Wang J, Ge J, Zheng F, Cao M, Dong Y, Liu D, Wang J, Lin Y, Du H, Gao GF, Wang X, Hu F, Tang J - PLoS ONE (2008)

Bottom Line: Bactericidal assays demonstrated that resistance of the mutant to polymorphonuclear leukocyte (PMN)-mediated killing was greatly decreased.Expression microarray analysis exhibited a transcription profile alteration of 26 various genes down-regulated in the DeltasalKR mutant.These findings suggest that SalK/SalR is requisite for the full virulence of ethnic Chinese isolates of highly pathogenic SS2, thus providing experimental evidence for the validity of this bioinformatically predicted PAI.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Third Military Medical University, Chongqing, China.

ABSTRACT

Background: Streptococcus suis serotype 2 (S. suis 2, SS2) has evolved into a highly infectious entity, which caused the two recent large-scale outbreaks of human SS2 epidemic in China, and is characterized by a toxic shock-like syndrome. However, the molecular pathogenesis of this new emerging pathogen is still poorly understood.

Methodology/principal findings: 89K is a newly predicted pathogenicity island (PAI) which is specific to Chinese epidemic strains isolated from these two SS2 outbreaks. Further bioinformatics analysis revealed a unique two-component signal transduction system (TCSTS) located in the candidate 89K PAI, which is orthologous to the SalK/SalR regulatory system of Streptococcus salivarius. Knockout of salKR eliminated the lethality of SS2 in experimental infection of piglets. Functional complementation of salKR into the isogenic mutant DeltasalKR restored its soaring pathogenicity. Colonization experiments showed that the DeltasalKR mutant could not colonize any susceptible tissue of piglets when administered alone. Bactericidal assays demonstrated that resistance of the mutant to polymorphonuclear leukocyte (PMN)-mediated killing was greatly decreased. Expression microarray analysis exhibited a transcription profile alteration of 26 various genes down-regulated in the DeltasalKR mutant.

Conclusions/significance: These findings suggest that SalK/SalR is requisite for the full virulence of ethnic Chinese isolates of highly pathogenic SS2, thus providing experimental evidence for the validity of this bioinformatically predicted PAI.

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Related in: MedlinePlus

Construction and confirmation analysis of the knockout mutant strain ΔsalKR.A), Strategy for deletion mutagenesis of salKR in S. suis 05ZYH33 by allelic replacement with a spectinomycin resistance cassette and schematic representation of the chromosomal structures before (left) and after (right) double cross-over (I) and single cross-over (II and III) recombination events between pUC::salKR and the chromosome of S. suis 05ZYH33. The location of the primers used in multiple-PCR detection are indicated by inverted arrowheads. The dotted lines represent the chromosomal sequences flanking the left and right arms of the construct. B, C and D), Southern hybridization analysis of the salKR region of S. suis wild type strain 05ZYH33 (lane 1), ΔsalKR mutant (lane 2), and a 3′ single cross-over mutant with pUC::salKR integrated into the chromosome of 05ZYH33 (lane 3). Genomic DNA from each strain was digested with Cla I and separated on 0.7% agarose gel. The hybridization probes used were as follows: SpcR gene (B), an internal fragment of salKR (C), and pUC18 (D). E), Multiple-PCR analysis of the ΔsalKR mutant. The primer combinations used in PCR are presented upon the lanes. Genomic DNA from the following strains were used as templates: wild type strain 05ZYH33 (lane 1, 4, 7, 10, 13, 16, 19 and 22), ΔsalKR mutant (lane 2, 5, 8, 11, 14, 17, 20 and 23) and the 3′ single cross-over mutant (lane 3, 6, 9, 12, 15, 18, 21 and 24). The 1 kb DNA ladder marker is shown to the left (M). Theoretical size (bp) of each of the PCR products generated with the primer combinations was shown in Table S2.
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pone-0002080-g003: Construction and confirmation analysis of the knockout mutant strain ΔsalKR.A), Strategy for deletion mutagenesis of salKR in S. suis 05ZYH33 by allelic replacement with a spectinomycin resistance cassette and schematic representation of the chromosomal structures before (left) and after (right) double cross-over (I) and single cross-over (II and III) recombination events between pUC::salKR and the chromosome of S. suis 05ZYH33. The location of the primers used in multiple-PCR detection are indicated by inverted arrowheads. The dotted lines represent the chromosomal sequences flanking the left and right arms of the construct. B, C and D), Southern hybridization analysis of the salKR region of S. suis wild type strain 05ZYH33 (lane 1), ΔsalKR mutant (lane 2), and a 3′ single cross-over mutant with pUC::salKR integrated into the chromosome of 05ZYH33 (lane 3). Genomic DNA from each strain was digested with Cla I and separated on 0.7% agarose gel. The hybridization probes used were as follows: SpcR gene (B), an internal fragment of salKR (C), and pUC18 (D). E), Multiple-PCR analysis of the ΔsalKR mutant. The primer combinations used in PCR are presented upon the lanes. Genomic DNA from the following strains were used as templates: wild type strain 05ZYH33 (lane 1, 4, 7, 10, 13, 16, 19 and 22), ΔsalKR mutant (lane 2, 5, 8, 11, 14, 17, 20 and 23) and the 3′ single cross-over mutant (lane 3, 6, 9, 12, 15, 18, 21 and 24). The 1 kb DNA ladder marker is shown to the left (M). Theoretical size (bp) of each of the PCR products generated with the primer combinations was shown in Table S2.

Mentions: To test the role of SalK/SalR in the pathogenesis of SS2, we constructed a homologous suicide plasmid, pUC::salKR with a SpcR cassette (Fig. 3A), and electrotransformed the competent cells of 05ZYH33. Positive transformants were first screened on THB agar plates under the selective pressure of spectinomycin. Colony PCR-based assays were performed to detect whether salKR was still present in the genome or not. One candidate mutant in which salKR failed to be amplified was obtained. To check this suspected mutant, Southern hybridization analyses were employed using probes of SpcR gene, an internal fragment of salKR, and pUC18, respectively. In wild type strain 05ZYH33, the internal fragment of salKR probe hybridized with a single 4.0 kb Cla I fragment (Fig. 3C, lane 1), whereas the SpcR and pUC18 probes did not hybridized with the genomic DNA (Fig. 3B and 3D, lane 1). In the suspected mutant, a 3.6 kb restriction fragment hybridized with the SpcR probe (Fig. 3B, lane 2), while no hybridization signal was detected when the internal fragment of salKR and pUC18 served as the probes (Fig. 3C and 3D, lane 2). In a single cross-over mutant which was set as the reference, both the SpcR and pUC18 probe hybridized with a 6.1 kb Cla I restriction fragment (Fig. 3B and 3D, lane 3), and the salKR probe hybridized with a 4.0 kb fragment (Fig. 3C, lane 3), indicating that this strain had pUC::salKR integrated into its chromosome by a single cross-over event at the 3′-flanking region of the salKR genes (Fig. 3A, III). Additionally, the allelic replacement of the wild-type salKR genes by the SpcR cassette in the suspected mutant was also confirmed by multiple-PCR analysis and sequencing with primers specific to genomic regions lying out the homologous left and right arms (Fig. 3A and 3E). All these results demonstrate that an isogenic knockout mutant of salKR (namely ΔsalKR) was successfully constructed. RT-PCR experiments later also showed that neither salK nor salR could be transcribed in ΔsalKR (data not shown), which further confirmed that salKR had been deleted from the bacterial chromosome.


SalK/SalR, a two-component signal transduction system, is essential for full virulence of highly invasive Streptococcus suis serotype 2.

Li M, Wang C, Feng Y, Pan X, Cheng G, Wang J, Ge J, Zheng F, Cao M, Dong Y, Liu D, Wang J, Lin Y, Du H, Gao GF, Wang X, Hu F, Tang J - PLoS ONE (2008)

Construction and confirmation analysis of the knockout mutant strain ΔsalKR.A), Strategy for deletion mutagenesis of salKR in S. suis 05ZYH33 by allelic replacement with a spectinomycin resistance cassette and schematic representation of the chromosomal structures before (left) and after (right) double cross-over (I) and single cross-over (II and III) recombination events between pUC::salKR and the chromosome of S. suis 05ZYH33. The location of the primers used in multiple-PCR detection are indicated by inverted arrowheads. The dotted lines represent the chromosomal sequences flanking the left and right arms of the construct. B, C and D), Southern hybridization analysis of the salKR region of S. suis wild type strain 05ZYH33 (lane 1), ΔsalKR mutant (lane 2), and a 3′ single cross-over mutant with pUC::salKR integrated into the chromosome of 05ZYH33 (lane 3). Genomic DNA from each strain was digested with Cla I and separated on 0.7% agarose gel. The hybridization probes used were as follows: SpcR gene (B), an internal fragment of salKR (C), and pUC18 (D). E), Multiple-PCR analysis of the ΔsalKR mutant. The primer combinations used in PCR are presented upon the lanes. Genomic DNA from the following strains were used as templates: wild type strain 05ZYH33 (lane 1, 4, 7, 10, 13, 16, 19 and 22), ΔsalKR mutant (lane 2, 5, 8, 11, 14, 17, 20 and 23) and the 3′ single cross-over mutant (lane 3, 6, 9, 12, 15, 18, 21 and 24). The 1 kb DNA ladder marker is shown to the left (M). Theoretical size (bp) of each of the PCR products generated with the primer combinations was shown in Table S2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2358977&req=5

pone-0002080-g003: Construction and confirmation analysis of the knockout mutant strain ΔsalKR.A), Strategy for deletion mutagenesis of salKR in S. suis 05ZYH33 by allelic replacement with a spectinomycin resistance cassette and schematic representation of the chromosomal structures before (left) and after (right) double cross-over (I) and single cross-over (II and III) recombination events between pUC::salKR and the chromosome of S. suis 05ZYH33. The location of the primers used in multiple-PCR detection are indicated by inverted arrowheads. The dotted lines represent the chromosomal sequences flanking the left and right arms of the construct. B, C and D), Southern hybridization analysis of the salKR region of S. suis wild type strain 05ZYH33 (lane 1), ΔsalKR mutant (lane 2), and a 3′ single cross-over mutant with pUC::salKR integrated into the chromosome of 05ZYH33 (lane 3). Genomic DNA from each strain was digested with Cla I and separated on 0.7% agarose gel. The hybridization probes used were as follows: SpcR gene (B), an internal fragment of salKR (C), and pUC18 (D). E), Multiple-PCR analysis of the ΔsalKR mutant. The primer combinations used in PCR are presented upon the lanes. Genomic DNA from the following strains were used as templates: wild type strain 05ZYH33 (lane 1, 4, 7, 10, 13, 16, 19 and 22), ΔsalKR mutant (lane 2, 5, 8, 11, 14, 17, 20 and 23) and the 3′ single cross-over mutant (lane 3, 6, 9, 12, 15, 18, 21 and 24). The 1 kb DNA ladder marker is shown to the left (M). Theoretical size (bp) of each of the PCR products generated with the primer combinations was shown in Table S2.
Mentions: To test the role of SalK/SalR in the pathogenesis of SS2, we constructed a homologous suicide plasmid, pUC::salKR with a SpcR cassette (Fig. 3A), and electrotransformed the competent cells of 05ZYH33. Positive transformants were first screened on THB agar plates under the selective pressure of spectinomycin. Colony PCR-based assays were performed to detect whether salKR was still present in the genome or not. One candidate mutant in which salKR failed to be amplified was obtained. To check this suspected mutant, Southern hybridization analyses were employed using probes of SpcR gene, an internal fragment of salKR, and pUC18, respectively. In wild type strain 05ZYH33, the internal fragment of salKR probe hybridized with a single 4.0 kb Cla I fragment (Fig. 3C, lane 1), whereas the SpcR and pUC18 probes did not hybridized with the genomic DNA (Fig. 3B and 3D, lane 1). In the suspected mutant, a 3.6 kb restriction fragment hybridized with the SpcR probe (Fig. 3B, lane 2), while no hybridization signal was detected when the internal fragment of salKR and pUC18 served as the probes (Fig. 3C and 3D, lane 2). In a single cross-over mutant which was set as the reference, both the SpcR and pUC18 probe hybridized with a 6.1 kb Cla I restriction fragment (Fig. 3B and 3D, lane 3), and the salKR probe hybridized with a 4.0 kb fragment (Fig. 3C, lane 3), indicating that this strain had pUC::salKR integrated into its chromosome by a single cross-over event at the 3′-flanking region of the salKR genes (Fig. 3A, III). Additionally, the allelic replacement of the wild-type salKR genes by the SpcR cassette in the suspected mutant was also confirmed by multiple-PCR analysis and sequencing with primers specific to genomic regions lying out the homologous left and right arms (Fig. 3A and 3E). All these results demonstrate that an isogenic knockout mutant of salKR (namely ΔsalKR) was successfully constructed. RT-PCR experiments later also showed that neither salK nor salR could be transcribed in ΔsalKR (data not shown), which further confirmed that salKR had been deleted from the bacterial chromosome.

Bottom Line: Bactericidal assays demonstrated that resistance of the mutant to polymorphonuclear leukocyte (PMN)-mediated killing was greatly decreased.Expression microarray analysis exhibited a transcription profile alteration of 26 various genes down-regulated in the DeltasalKR mutant.These findings suggest that SalK/SalR is requisite for the full virulence of ethnic Chinese isolates of highly pathogenic SS2, thus providing experimental evidence for the validity of this bioinformatically predicted PAI.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Third Military Medical University, Chongqing, China.

ABSTRACT

Background: Streptococcus suis serotype 2 (S. suis 2, SS2) has evolved into a highly infectious entity, which caused the two recent large-scale outbreaks of human SS2 epidemic in China, and is characterized by a toxic shock-like syndrome. However, the molecular pathogenesis of this new emerging pathogen is still poorly understood.

Methodology/principal findings: 89K is a newly predicted pathogenicity island (PAI) which is specific to Chinese epidemic strains isolated from these two SS2 outbreaks. Further bioinformatics analysis revealed a unique two-component signal transduction system (TCSTS) located in the candidate 89K PAI, which is orthologous to the SalK/SalR regulatory system of Streptococcus salivarius. Knockout of salKR eliminated the lethality of SS2 in experimental infection of piglets. Functional complementation of salKR into the isogenic mutant DeltasalKR restored its soaring pathogenicity. Colonization experiments showed that the DeltasalKR mutant could not colonize any susceptible tissue of piglets when administered alone. Bactericidal assays demonstrated that resistance of the mutant to polymorphonuclear leukocyte (PMN)-mediated killing was greatly decreased. Expression microarray analysis exhibited a transcription profile alteration of 26 various genes down-regulated in the DeltasalKR mutant.

Conclusions/significance: These findings suggest that SalK/SalR is requisite for the full virulence of ethnic Chinese isolates of highly pathogenic SS2, thus providing experimental evidence for the validity of this bioinformatically predicted PAI.

Show MeSH
Related in: MedlinePlus