| Streptococcus suis serotype 2 (S. suis 2) is an important worldwide zoonotic pathogen associated with a wide range of diseases in pigs, including septicaemia, meningitis, pneumonia, endocarditis , arthritis and even sudden death. Occasionally, S. suis 2 can also be transmitted to human beings through respiratory tract or wound contamination,posing the serious threat to the pig-breeding industry and the correlation jobholders. Furthermore, two major emerging infectious disease outbreaks of S. suis 2 in China (one in Jiangsu Province, 1998, and the other in Sichuan Province, 2005), raised considerable international concerns among the public health professionals. A key feature of these two Chinese outbreaks is the prevalence of streptococcal toxic shock syndrome (STSS) manifesting itself as acute high fever, multiple organ failures, short course of disease and high lethality (62.7%~81.3%).But the concrete pathogenesis mechanism still not clear.Two-component signal transduction systems (TCSTS) composed of a membrane-bound sensor(histidine kinase, HK) and a cytoplasmic response regulator (RR), is an important mechanism used by bacteria to sense and response to environmental stimuli, can regulate many kinds of toxicity factor expression to complete its pathogenesis process. Our joint research group completed a comprehensive study of comparative genomics, decoding the whole genome sequences of the virulent S. suis 2 strain 05ZYH33 ( isolated from Chinese STSS patients), and found that there are 11 TCSTS and 5 orphan response regulators in it.△We focused on a unique TCSTS named 1094HK/1095RR according to its code gene position, discovered that 1095RR and the CiaR amino acid sequence of many kinds of streptococcus (S. pneumonia, S. pyogenes, S.mutants and so on) uniformity reaches as high as above 80%. The system CiaRH of S. pneumonia has been implicated in maintenance of cell integrity, competence and virulence, but the reports of S. suis regarding the CiaRH research have not been emerged.In this study, the ciaR gene deletion mutant of the wild type strain 05ZYH33 (namely△ciaR) was screened by homologous recombination, PCR analysis and Southern hybridization were used to confirm its correction. After repeatedly serial subcultivation, the spectinomycin resistance phenotype was found to be stable in the△ciaR. Through compared under the same conditions, colony and cell morphology, hemolytic activity were examined but no significant differences between the wild type and mutant strain could be ascertained. The△ciaR 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 40mM H2O2 for 15min.Viable cells were counted by plating them onto THB agar plates and the results were expressed as percentage of survival. The△ciaR 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 condition the△ciaR mutant presented growth and survival defects. Put bacterial collection of stationary phases(OD600=0.6～0.8) by centrifuge to 0.1%Triton X-100 to shaking culture, and fast autolysis of the△ciaR was observed by detect OD600 every one hour. Nine of ten mice which infected with the wild type or mutant(1×109CFU/mouse) died at 16hours post-infection. To sum up, lack of the ciaR gene reduces the ability of S. suis 2 05ZYH33 proliferation, tolerance to the oxidative stresses, acid environment and 0.1% Triton X-100 in vitro, but no differences of virulence to mice.By bioinformatics analysis we knew that the CiaR protein is the DNA-binding protein, in order to identify genes which were regulated, to express the recombinant protein in prokaryotic cells at first. The titer of polyclonal antibody from rabbit serum immunized with recombinant protein reached 1:204800, and the serum could be specifically reacted with the recombinant protein demonstrated by Western blot. All above work lay the foundation for further developing the CiaR regulative mechanism research. |
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