Studies On Regulation Of Transcription Factor Cody To The Virulence Of Streptococcus Suis Serotype2

Streptococcus suis serotype2(SS2) is one of the most important swine pathogens, and an emerging, life-threatening zoonotic agent in both pigs and humans.. In recent years, many researchers pay more and more attention on the pathogenic of Streptococcus suis serotype2. With the exception of some of the traditional known virulence factors such as capsular polysaccharide, hemolysin, and extracellular factor, many new virulence factors have been confirmed such as two-component regulation system Sa1K-Sa1R, CiaRH, orphans regulation factor CovR, endothelin-converting enzyme SsPep and so on. Although many scholars interpreted the Streptococcus suis serotype2pathogenesis from different perspectives, SS2numerous virulence factors are synergy. Therefore, it is difficult to explain fully its pathogenesis only by a single virulence factor. In fact, the most efficient way is through a large number of transcription factors interactions, the formation of a sophisticated global regulatory network to control the expression of the bacterial genome.At present, Streptococcus pyogenes the global regulatory network has been in-depth understanding, but the global regulatory network for SS2is still very limited. CodY is an important global transcription regulator in most gram-positive bacteria, and regulates the expression of many virulence genes, thus affecting the pathogen pathogenicity. The gene has been studied deeply in Streptococcus pyogenes, Staphylococcus aureus, Streptococcus pneumoniae and so on, but no reports about codY in Streptococcus suis type2. This study identified a transcription regulator CodY in China high virulent strain SS205ZYH33, constructed codY gene mutants to study mutants a series of reseach about the basic biological characteristics. By microarray get the differentially expressed genes, which migth be regulated by CodY, and verification by realtime qPCR. Analysis of the CodY possible regulation mechanism in SS2, which can provide a theory foundation for elucidating SS2molecular pathogenesis, and provide new ideas as well as the design of new vaccines and drug targets for Streptococcus suis. Specific experiment content as follows:1. Construction and identification of the transcriptional regulation factor CodY mutant AcodY in SS2Reference to Streptococcus suis serotype205ZYH33whole genome sequences, design primers, Streptococcus suis serotype2Sichuan isolates (SC19) genomic DNA as template, amplification of the the gene codY internal section of gene codY, which did not include the start codon and function domain (Helix-Turn-Helix domain, HTH). Use the temperature-sensitive the suicide plasmid pSET4s, and construction the recombinant plasmid pSET4s-codY. The recombinant plasmid pSET4s-codY’ was transformed into competent cells of the wild type strain SC19, while the use of spectinomycin and temperature screening to the mutant. Using PCR, RT-PCR, sequencing and western blot to confirm that the mutant codY was constructed successfully, and named it AcodY.2. Research on the basic biological characteristics of△codY mutant in SS2In our work, the mutant AcodY phenotype, the growth curve determination, the observation of the morphology, the detection of hemolytic, cell infection experiment, mice the median lethal dose calculation, organizations carrier experiment and so on were researched. The results showed that the growth rate of AcodY slowed down obviously, and declined adhesion ability to HEp-2cell, while AcodY was more likely killed by PMN and macrophages; that the capsules of the mutant AcodY were thinner and more porous than that of wild type strain. The bacterial hemolytic activity of the mutant AcodY was weakened. Besides, the results of the median lethal dose experiment showed that the mutant AcodY virulence was attenuated5.68fold than the wild-type strain. The quantity of pathogen were measured at various time points in the heart, lungs, brain, blood contaminated amount, and the various organs of the mutant carrier were lower than the wild type strain. The mutant AcodY was cleared easily. In summary, the mutant AcodY virulence declined.3. The research on the regulation mechanism and pathogenesis of CodY in SS2The microarray data showed that the absence of CodY led to downregulated of164genes foldchange more than2, including virulence-associated genes, and capsular synthesis genes; and214genes were positively regulated in the entire2178genes, including a variety of amino acid synthesis genes, nitrogen metabolism-related genes and so on. Select the part of the virulence genes to investigate in order to verify the reliability of the microarray data by realtime qPCR. The CodY regulation situation was further investigateed by electrophoretic mobility shift assays (EMSA). The results showed that some virulence-related genes could be regulated directly by CodY, thereby affecting the bacterial virulence.