【作者】 冯晓丹；

【导师】 唐家琪；

【作者基本信息】 南京医科大学 ， 病原生物学， 2009， 硕士

【摘要】 猪链球菌2型(Streptococcus suis serotype 2)是一种呈全球分布的人兽共患病病原体。S. suis 2感染不仅可致猪急性败血症、脑膜炎、关节炎、心内膜炎、关节炎及急性死亡,并且可通过伤口和呼吸道等传播途径,导致人的感染发病和死亡,对养猪业及相关从业人员均造成严重威胁。1998年和2005年我国江苏省和四川省分别暴发大规模S. suis 2感染疫情,患者出现链球菌中毒性休克综合征(Streptococcal toxic shock syndrome, STSS),病情凶险,病死率高(62.7%～81.3%),具体致病机制尚不清楚。二元信号转导系统(two-component signal transduction systems,TCSTS)是广泛存在于细菌中的调控单元,由膜上的组氨酸激酶(histidine kinase, HK)和胞内的反应调节因子(response regulator, RR)两部分组成,在致病菌感染宿主的过程中能调控多种毒力因子的表达以完成其致病过程。本课题组完成了S. suis 2强毒株05ZYH33(分离自STSS病人)的全基因组测序,并对基因组进行注释。结果显示,05ZYH33中存在11个TCSTS和5个孤儿反应调控因子,根据其编码基因所在阅读框的位置,选取其中一个TCSTS,命名为1094HK/1095RR,同源性比对发现1095RR与多种链球菌(肺炎链球菌、化脓链球菌、变形链球菌等)的CiaR氨基酸序列一致性高达80%以上。现已证实在肺炎链球菌中,CiaRH系统对维持细菌的完整性、感受性、毒性等生物活性发挥重要作用,而在猪链球菌中对于CiaRH的研究国内外尚未见报道。本课题以S. suis 2强毒株05ZYH33为对象,基于同源重组原理筛选获得ciaR基因缺失株,PCR分析及Southern杂交结果证实突变株构建成功。经过反复传代培养,证明敲除株可以稳定生长,壮观霉素抗性表型能够在敲除株中稳定表达。同时在相同条件下观察发现敲除株与野生株的菌落形态和溶血活性均无明显差别。通过绘制生长曲线,发现与野生株相比,敲除株在37℃和40℃其OD600值均明显降低;与40mmol/L过氧化氢共作用15min后涂板计数,突变株存活率明显低于野生株;不同pH值的培养基中培养,发现在pH5.0的酸性环境中突变株OD600值明显低于野生株;收集稳定期(OD600=0.6～0.8)细菌的菌体,置于含0.1% Triton X-100的液体中振荡培养诱导细菌自溶,每间隔1h检测发现突变株OD600值明显低于野生株;野生株与突变株对BALB/c小鼠攻毒(约1×109CFU/只),各10只,两组16h后均死亡9只。综合以上实验结果说明敲除了05ZYH33菌株中的ciaR基因,细菌的生长减慢、繁殖能力减弱,抗氧化应激能力下降,在酸性环境中生长存活能力下降,0.1% Triton X-100诱导下自溶能力增强;但对小鼠的毒力未见明显差别。生物信息学分析发现CiaR蛋白为DNA结合蛋白,为了明确其调控的具体基因,较全面的诠释该调控系统的生物学功能,首先需要获得纯化的CiaR蛋白。本文通过原核表达系统体外表达了CiaR融合蛋白,将纯化的重组蛋白加免疫佐剂后免疫新西兰兔,获得高效价(1:204800)的兔抗CiaR多克隆抗体血清。Western blot结果显示,该血清可与CiaR重组蛋白发生特异性反应,说明该融合蛋白具有良好的免疫原性。所有上述工作,为进一步开展CiaR的调控机理研究奠定了基础。更多还原

【Abstract】 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.更多还原