Xiuzhen Pan

Nanjing Normal University

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Jiaqi Tang

Northeast Agricultural University

Changjun Wang

Guangdong Academy of Medical Sciences

Zheng Feng

Chengdu University of Technology

Youjun Feng

Shenzhen Third People’s Hospital

Xianfu Li

Dali University

Dan Hu

Second Affiliated Hospital & Yuying Children's Hospital of Wenzhou Medical University

Zhu‐Qing Shao

Nanjing University

Min Cao

Nanjing Tech University

Changjun Wang

Anhui Medical University

Junchao Ge

Nanjing General Hospital of Nanjing Military Command

Cooperative Institutions

Chinese Academy of Sciences

Nanjing General Hospital of Nanjing Military Command

Shanghai Jiao Tong University

Nanjing Medical University

Huadong Hospital

Fudan University

Nanjing Normal University

University of Chinese Academy of Sciences

Nanjing Agricultural University

Chinese Academy of Medical Sciences & Peking Union Medical College

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Identification of the targets of CovR by chromatin immunoprecipitation

Yixue yanjiusheng xuebao (2013)

Xiuzhen Pan

Objective CovR plays a very important role in regulating the expression of virulence factors and inducing the pathogenicity of streptococcus suis.This study aimed to further investigate the regulating mechanism of CovR using chromatin immunoprecipitation to screen and identify the downstream targets of CovR.Methods Living cells of streptococcus suis serotype 2 05ZYH33 were fixed with formaldehyde,sonicated to shear the DNA into 100-500 bp fragments and then immunoprecipitated with the anti-CovR monoclonal antibody.The DNA fragments were extracted and purified,and the primers of 8 potential target gene promoter regions were designed.Specific amplification of DNA regions by each primer set was checked by PCR reaction using the purified DNA as the template.Results The optimal formaldehyde concentration to achieve efficient and reversible cross-linking turned out to be 1%.DNA fragments of 100-500 bp were obtained using 40 rounds of 10 seconds each at the power of 60W.The CovR binding sequences on the promoters of the gene SSU1668 and SSU1732 were found in the anti-CovR monoclonal antibody immunoprecipitated DNA fragments,while another 6 gene promoter regions showed no positive bands.Conclusion SSU1668 and SSU1732 gene promoter regions exist in the anti-CovR monoclonal antibody immunoprecipitated DNA fragments,which shows that the SSU1668 and SSU1732 genes are CovR target genes.

Chromatin immunoprecipitation

Immunoprecipitation

Primer (cosmetics)

Streptococcus Pyogenes

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Construction and characterization of a two-component signal transduction system ciaR gene knock-out of Streptococcus suis serotype 2.

Zhongguo renshougonghuanbing zazhi (2009)

Xiaodan Feng Cheng Gong Jing Wang Changjun Wang Xiuzhen Pan

To construct ciaR gene deletion strain(ΔciaR)which encodes the response regulator of the two-component signal transduction system 1094HK/1095RR in Streptococcus suis serotype 2 virulent strain 05ZYH33,and to investigate biological functions of the ΔciaR.Recombinant gene knock-out vector was constructed consisting of SpcR cassette with flanking homology regions to the ciaR gene,and the ciaR gene deletion mutant was screened by homologous recombination.The ΔciaR mutant was confirmed by PCR analysis and Southern hybridization.The ΔciaR mutant was found to grow more slowly than the wild type strain at 37℃ and 40℃,as judged by OD600.The sensitivity of cells to peroxide was test by the exposure of aliquots of cultures to 40mmol/L H2O2 for 15min.Viable cells were counted by plating them onto THB agar plates and the results were expressed as percentage of survival.The ΔciaR strain was significantly more sensitive to peroxide than the wild type.No obvious differences were observed at different pHs except at pH5.0,in which the ΔciaR mutant presented growth and survival defects.Nine of ten mice infected with the wild type or mutant(1×109CFU/mouse)died at 16hours post-infection.It is evident that lack of the ciaR gene reduces the proliferative ability of S.suis 2 05ZYH33,tolerance to the oxidative stresses and survival at acid environment in vitro,but no differences of virulence to mice.

Response regulator

Wild type

Streptococcus suis

Streptococcus oralis

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A Multiplex PCR Assay for Simultaneous Detection of Six Serotypes of Streptococcus suis

Xiuzhen Pan

A multiplex PCR assay for detection and identification of various streptococcus suis strains in tonsillar specimens from pigs was developed and evaluated.In this research five distinct DNA targets were chose: One target,based on the gdh gene,encoded the glutamate dehydrogenase of streptococcus suis,can be applied to the detection of streptococcus suis at species level.The other targets,based on SS capsular polysaccharide(cps) genes specific for serotypes 1(and 14),2(and 1/2),7,9 were amplified by multiplex PCR.73 isolates,which included 49 strains of streptococcus suis and 24 strains of negative control,and 94 clinical specimens(45 clinical strains in Sichuan)were detected by the multiplex PCR assay.In 73 streptococcus suis strains,64(87.5%)strains were gdh+.There were no PCR products in strains of serotypes 13,19,30,32,33,34.The multiplex PCR could detect all strains of serotypes 1(and 14),2(and 1/2),7,9.In 45 clinical strains in Sichuan,the PCR results of 41 strains were gdh+cps2+.These results indicated that the PCR can be used as a reliable species-specific molecular diagnostic reagent and a serotype-specific method for the detection of streptococcus suis and six main serotypes 1,2,1/2,7,9 and 14,respectively.The PCR method has potential value for clinical and epidemiological applications.

Streptococcus suis

Multiplex

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THE ROLE SURFACE PROTEIN PLAY ENOLASE IN THE SEVERE INFECTION OF STREPTOCOCCUS SUIS SEROTYPE 2

Journal of Jinggangshan University (2010)

Xiuzhen Pan

Objective: To explore the role of enolase from Streptococcus suis serotype 2 (S. suis 2) 05ZYH33 in the bacterial adherence to host cells and the suppression of host immune response. Methods: This study used the methods of flow cytometry(FCM), cell adhension assay of Hep 2 cells and hemolytic antibody plaques formation test. Results: The results of FCM showed that Enolase located on the surface of 05ZYH33, the results of adherence assay of Hep 2 cells indicated the enolase was involed in the bacterial adherence to host cells, the hemolytic antibody plaques formation test results indicated that enolase played a role in the suppression of the specific host immune response. Conclusion: As a surface protein, enolase played pivotal roles in the severe infection of S. suis 2.

Enolase

Streptococcus suis

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SalK/SalR, a Two-Component Signal Transduction System, Is Essential for Full Virulence of Highly Invasive Streptococcus suis Serotype 2

PLoS ONE (2008)

Ming Li Changjun Wang Youjun Feng Xiuzhen Pan Gong Cheng

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.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.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.

Streptococcus suis

Pathogenicity island

Human pathogen

10.1371/journal.pone.0002080

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Development of reverse transcription loop-mediated isothermal amplification assays to detect Hantaan virus and Seoul virus

Journal of Virological Methods (2015)

Dan Hu Lina Hao Jinhai Zhang Pingping Yao Qi Zhang

Hantaan virus

10.1016/j.jviromet.2015.04.017

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Prokaryotic expression of hemolysin-like protein gene in highly virulent strains of Streptococcus suis serotype 2 and preparation of the polyclonal antibodies against its expressed products.

Zhongguo renshougonghuanbing zazhi (2009)

Mingyue Han Xiuzhen Pan Zhu‐Qing Shao Junchao Ge Changjun Wang

To express the gene encoding hemolysin-like protein(HLP)in highly virulent strains of Streptococcus suis serotype 2 and to prepare the polyclonal antibodies against its expressed products,the hlp gene from the genomic DNA in the virulent strain 05ZYH33 was amplified by PCR using a pair of specific primers and subcloned into plasmid pMD18-T and pET32a with double digestion of BamHⅠ and XhoI.Subsequently,the prokaryotic expression plasmid pET32a::hlp was transformed to E.coli BL21(DE3)after identification by restriction endonuclease digestion and DNA sequencing.Upon induction with IPTG,E.coli BL21 containing the recombinant plasmid could express a distinct band with a molecular weight of 53 kDa,which was similar to the predicted band of HLP protein as demonstrated by SDS-PAGE.The recombinant HLP protein was purified by Ni2+-NTA affinity chromatography and the recombinant protein was used as antigen to immunize New Zealand rabbits to prepare the polyclonal antibodies.Titers of the antibodies produced in the immunized rabbits were determined by indirect ELISA and Western blot assay.It was demonstrated that the constructed plasmid could be highly expressed in the host bacteria and the recombinant protein with high purity was obtained after His affinity chromatography.Sera of the immunized rabbits could react specifically with the purified recombinant HLP protein as demonstrated by ELISA and Western blot assay with a titer of polyclonal antibodies of 1∶409600.This recombinant obtained and the polyclonal antibodies produced would be useful in the further studies on the hlp gene.

Polyclonal antibodies

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Experimental Studies on Serial Passage of HAV in Human Leukocytes and Propagation of HAV In Various Kinds of Leukocytes in Vitro

Virologica Sinica (1991)

Jishan Zheng LI Fa-qing Lin Jian-Men Xiuzhen Pan Tan Wei-guo

Ficoll

Human blood

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Streptococcus suisEnolase Functions as a Protective Antigen Displayed on the Bacterial Cell Surface

The Journal of Infectious Diseases (2009)

Youjun Feng Xiuzhen Pan Wen Sun Changjun Wang Huimin Zhang

Streptococcus suis serotype 2 (SS2) has evolved into a highly infectious entity, posing a great threat to public health. Screening for and identification of protective antigens plays an important role in developing therapies against SS2 infections.Multiple strategies were used to investigate a new surface protein that has the potential to be a protective antigen. These strategies included molecular cloning, biochemical and biophysical analyses, enzymatic assay, immunological approaches (eg, immunoelectron microscopy), and experimental infections of animals.We identified an enolase gene from SS2 and systematically characterized its protein product, enolase. Biophysical data indicated that S. suis enolase is an octameric protein. Enzymatic assays verified its ability to catalyze the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate. In consideration of the strong antigenicity of enolase, an efficient enolase-based method was established for monitoring SS2 infections. Combined evidence strongly indicated that SS2 enolase can localize on the bacterial cell surface and facilitate bacterial adherence. Additionally, we found that enolase can confer complete protection against SS2 infection to mice, which suggests that enolase has potential as a vaccine candidate.We conclude that S. suis enolase functions as a protective antigen displayed on the bacterial cell surface and that it can be used to develop new strategies to combat SS2 infections.

Enolase

Streptococcus suis

Antigenicity

10.1086/644602

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Isolation and characterization of a native avirulent strain of Streptococcus suis serotype 2: a perspective for vaccine development

Scientific Reports (2015)

Xinyue Yao Ming Li Jing Wang Changjun Wang Dan Hu

Abstract Streptococcus suis , an emerging infectious pathogen, is the cause of two large-scale outbreaks of human streptococcal toxic shock syndrome in China and has attracted much attention from the scientific community. The genetic basis of its pathogenesis remains enigmatic and no effective prevention measures have been established. To better understand the virulence differentiation of S. suis and develop a promising vaccine, we isolated and sequenced a native avirulent S. suis strain (05HAS68). Animal experiments revealed that 05HAS68 is an avirulent strain and could protect piglets from the attack of virulent strains. Comparative genomics analyses demonstrated the genetic basis for the lack of virulence in 05HAS68, which is characterized by the absence of some important virulence-associated factors and the intact 89K pathogenicity island. Lack of virulence was also illustrated by reduced survival of 05HAS68 compared to a virulent strain in pig whole blood. Further investigations revealed a large-scale genomic rearrangement in 05HAS68, which was proposed to be mediated by transposase genes and/or prophages. This genomic rearrangement may have caused the genomic diversity of S. suis and resulted in biological discrepancies between 05HAS68 and highly virulent S. suis strains.

Streptococcus suis

Isolation

Strain (injury)

10.1038/srep09835

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Citations (22)