Image1.JPEG

<p>Streptococcus suis serotype 2 is an important swine pathogen and an emerging zoonotic agent that causes severe infections. Recent studies have reported a eukaryotic-like Ser/Thr protein kinase (STK) gene and characterized its role in the growth and virulence of different S. suis 2 strains. In the present study, phosphoproteomic analysis was adopted to identify substrates of the STK protein. Seven proteins that were annotated to participate in different cell processes were identified as potential substrates, which suggests the pleiotropic effects of stk on S. suis 2 by targeting multiple pathways. Among them, a protein characterized as cell division initiation protein (DivIVA) was further investigated. In vitro analysis demonstrated that the recombinant STK protein directly phosphorylates threonine at amino acid position 199 (Thr-199) of DivIVA. This effect could be completely abolished by the T199A mutation. To determine the specific role of DivIVA in growth and division, a divIVA mutant was constructed. The ΔdivIVA strain exhibited impaired growth and division, including lower viability, enlarged cell mass, asymmetrical division caused by aberrant septum, and extremely weak pathogenicity in a mouse infection model. Collectively, our results reveal that STK regulates the cell growth and virulence of S. suis 2 by targeting substrates that are involved in different biological pathways. The inactivation of DivIVA leads to severe defects in cell division and strongly attenuates pathogenicity, thereby indicating its potential as a molecular drug target against S. suis.</p

Table1.XLS

<p>Streptococcus suis serotype 2 is an important swine pathogen and an emerging zoonotic agent that causes severe infections. Recent studies have reported a eukaryotic-like Ser/Thr protein kinase (STK) gene and characterized its role in the growth and virulence of different S. suis 2 strains. In the present study, phosphoproteomic analysis was adopted to identify substrates of the STK protein. Seven proteins that were annotated to participate in different cell processes were identified as potential substrates, which suggests the pleiotropic effects of stk on S. suis 2 by targeting multiple pathways. Among them, a protein characterized as cell division initiation protein (DivIVA) was further investigated. In vitro analysis demonstrated that the recombinant STK protein directly phosphorylates threonine at amino acid position 199 (Thr-199) of DivIVA. This effect could be completely abolished by the T199A mutation. To determine the specific role of DivIVA in growth and division, a divIVA mutant was constructed. The ΔdivIVA strain exhibited impaired growth and division, including lower viability, enlarged cell mass, asymmetrical division caused by aberrant septum, and extremely weak pathogenicity in a mouse infection model. Collectively, our results reveal that STK regulates the cell growth and virulence of S. suis 2 by targeting substrates that are involved in different biological pathways. The inactivation of DivIVA leads to severe defects in cell division and strongly attenuates pathogenicity, thereby indicating its potential as a molecular drug target against S. suis.</p

Image3.JPEG

<p>Streptococcus suis serotype 2 is an important swine pathogen and an emerging zoonotic agent that causes severe infections. Recent studies have reported a eukaryotic-like Ser/Thr protein kinase (STK) gene and characterized its role in the growth and virulence of different S. suis 2 strains. In the present study, phosphoproteomic analysis was adopted to identify substrates of the STK protein. Seven proteins that were annotated to participate in different cell processes were identified as potential substrates, which suggests the pleiotropic effects of stk on S. suis 2 by targeting multiple pathways. Among them, a protein characterized as cell division initiation protein (DivIVA) was further investigated. In vitro analysis demonstrated that the recombinant STK protein directly phosphorylates threonine at amino acid position 199 (Thr-199) of DivIVA. This effect could be completely abolished by the T199A mutation. To determine the specific role of DivIVA in growth and division, a divIVA mutant was constructed. The ΔdivIVA strain exhibited impaired growth and division, including lower viability, enlarged cell mass, asymmetrical division caused by aberrant septum, and extremely weak pathogenicity in a mouse infection model. Collectively, our results reveal that STK regulates the cell growth and virulence of S. suis 2 by targeting substrates that are involved in different biological pathways. The inactivation of DivIVA leads to severe defects in cell division and strongly attenuates pathogenicity, thereby indicating its potential as a molecular drug target against S. suis.</p

Image2.JPEG

<p>Streptococcus suis serotype 2 is an important swine pathogen and an emerging zoonotic agent that causes severe infections. Recent studies have reported a eukaryotic-like Ser/Thr protein kinase (STK) gene and characterized its role in the growth and virulence of different S. suis 2 strains. In the present study, phosphoproteomic analysis was adopted to identify substrates of the STK protein. Seven proteins that were annotated to participate in different cell processes were identified as potential substrates, which suggests the pleiotropic effects of stk on S. suis 2 by targeting multiple pathways. Among them, a protein characterized as cell division initiation protein (DivIVA) was further investigated. In vitro analysis demonstrated that the recombinant STK protein directly phosphorylates threonine at amino acid position 199 (Thr-199) of DivIVA. This effect could be completely abolished by the T199A mutation. To determine the specific role of DivIVA in growth and division, a divIVA mutant was constructed. The ΔdivIVA strain exhibited impaired growth and division, including lower viability, enlarged cell mass, asymmetrical division caused by aberrant septum, and extremely weak pathogenicity in a mouse infection model. Collectively, our results reveal that STK regulates the cell growth and virulence of S. suis 2 by targeting substrates that are involved in different biological pathways. The inactivation of DivIVA leads to severe defects in cell division and strongly attenuates pathogenicity, thereby indicating its potential as a molecular drug target against S. suis.</p

Table2.DOC

<p>Streptococcus suis serotype 2 is an important swine pathogen and an emerging zoonotic agent that causes severe infections. Recent studies have reported a eukaryotic-like Ser/Thr protein kinase (STK) gene and characterized its role in the growth and virulence of different S. suis 2 strains. In the present study, phosphoproteomic analysis was adopted to identify substrates of the STK protein. Seven proteins that were annotated to participate in different cell processes were identified as potential substrates, which suggests the pleiotropic effects of stk on S. suis 2 by targeting multiple pathways. Among them, a protein characterized as cell division initiation protein (DivIVA) was further investigated. In vitro analysis demonstrated that the recombinant STK protein directly phosphorylates threonine at amino acid position 199 (Thr-199) of DivIVA. This effect could be completely abolished by the T199A mutation. To determine the specific role of DivIVA in growth and division, a divIVA mutant was constructed. The ΔdivIVA strain exhibited impaired growth and division, including lower viability, enlarged cell mass, asymmetrical division caused by aberrant septum, and extremely weak pathogenicity in a mouse infection model. Collectively, our results reveal that STK regulates the cell growth and virulence of S. suis 2 by targeting substrates that are involved in different biological pathways. The inactivation of DivIVA leads to severe defects in cell division and strongly attenuates pathogenicity, thereby indicating its potential as a molecular drug target against S. suis.</p

Phylogenetic Trees of Six Representative Isolates Based on Comparison of 16S rDNA and Five Putative Virulence-Associated-Factor Genes with Known Sequences

<p>Swine isolates from Sichuan ( S. suis ZYS3 and S. suis ZYS8) labeled in green, human isolates ( S. suis ZYH13 and S. suis ZYH14) from Sichuan labeled in red, Jiangsu isolates from 1998 ( S. suis 9801 and S. suis Habb) labeled in blue, and the standard highly virulent strain S. suis P1/7 labeled in pink. All representative strains from other streptococcus species or isolates of S. suis 2 are as indicated in the tree. </p

Microscopic Characterization of Sectioned Liver Tissue from Patients Who Had Died

<div><p>(A) Light image of a liver tissue section (×100). The central vein is indicated with an arrow.</p> <p>(B) Light image of a liver tissue section (×200).</p> <p>(C) The convergent zone is indicated with an arrow (×100).</p> <p>(D) TEM image of a liver tissue section (×20,000). A bacterium found in the tissue is highlighted with an arrow.</p></div

RFLP Analysis of Different S. suis 2 Isolates

<p> S. suis S10: a highly virulent strain from China; <i>S</i>. <i>suis</i> 9801: swine isolate from Jiangsu Province in 1998; S. suis Habb: human isolate from Jiangsu Province in 1998; S. suis ZYS3: swine isolate from Sichuan Province in 2005; S. suis ZYH13: human isolate from Sichuan Province in 2005; M: 1 kb DNA Ladder (MBI Ferments, Gdansk, Poland). </p

Immunohistochemical Analysis of Liver Tissue Sections from Dead Patients Incubated with Normal and S. suis 2-Infected Swine Serum

<div><p>(A) Staining with normal swine serum detected no S. suis 2 antigen. </p> <p>(B) Staining with the serum from the infected swine indicated the presence of S. suis 2 antigen. </p></div

Detection of the Pathogenic SS2 and Identification of Its Specific Genes

<div><p>(A) Light microscopy image of the isolates cultured from autopsy specimens.</p> <p>GP⁺ cocci (pointed to with black arrows) are arranged in various short chains (×100). </p> <p>(B) Qualitative PCR detection of isolates from the liver of fatal human cases with a set of primers specific for <i>S</i>. <i>suis</i> 2. M: 100bp DNA marker (Fermentas, Vilnius, Lithuania). CK: 16S rDNA PCR product from the R 735 standard strain of S. suis 2. Multi-PCR: performed with a set of unique primers specific for <i>mrp, epf, suilysin,</i> and <i>cps-2J,</i> respectively. </p></div