【作者】 袁芳艳；

【导师】 陈焕春； 贝为成；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2010， 博士

【摘要】 猪链球菌病是由猪链球菌(Streptococcus suis,S.suis)引起的一种当前严重危害我国养猪业的重要细菌性传染病。根据荚膜多糖抗原成分不同,猪链球菌分33个血清型(1-31、33、35型和1/2型)。其中猪链球菌2型(Streptococcus suis serotype 2,SS2)作为一种重要人畜共患传染病病原,不仅影响着养殖业的健康发展,还严重危害着人类的健康,引起了社会的高度关注。近几年,SS2已成为国内外研究的热点,尤其是关于其分子致病与免疫机理方面的研究。病原微生物致病由多种毒力因子的协同作用而引起,这些毒力因子受某些调控系统的调控,形成一个复杂的调控网络。双组分调控系统(Two-Component Regulatory System,TCS)是广泛存在于细菌中的一种信号转导机制。它首先感应外界环境的变化,继而把信息传给内部系统,导致细菌毒力因子等表达发生变化。大量研究证实,TCS参与调控细菌致病性、生长代谢、营养代谢、耐药性及毒力因子的表达等多种生物学功能。随着我国猪链球2型05ZYH33和98HAH12菌株全基因组测序的完成,在SS2发现至少有15对双组分调控系统。然而,在这些调控系统仅SalK-SalR和两个“孤儿”调控因子RevS.CovR被研究报道。本论文选取了其中一对双组分调控系统1910HK/RR为研究对象,开展了一系列生物学研究,以探讨其与SS2野生菌株SC19(WT)致病性的关系。主要研究内容包括：1.基因缺失突变株Δ1910hk/rr和互补菌株CΔ1910hk/rr的构建及鉴定参考SS2强致病株05ZYH33全基因组序列,设计引物,以四川分离的WT为亲本株,提取其基因组DNA为模板,PCR分别扩增基因1910hk/rr上下游同源臂、开放阅读框(ORF)和全长序列,利用温度敏感型“自杀性”质粒pSET4s,构建重组转移质粒pSET4s-1910hk/rr。将重组转移质粒电转化到WT感受态细胞中,通过28℃和37℃温度双标记和抗生素筛选,初步获得1910hk/rr基因缺失菌株。应用PCR、RT-PCR和测序对突变株进一步鉴定,成功构建基因缺失突变株Δ1910hk/rr。利用穿梭质粒pAT18,将所缺失的基因1910hk/rr及其启动子序列一起转入相应的基因缺失突变菌株Δ1910hk/rr中,通过PCR和RT-PCR鉴定,获得了相应的互补菌株CΔ1910hk/rr。2.WT、Δ1910hk/rr和CΔ1910hk/rr的体外生物学特性比较研究在体外相同培养条件下,开展了WT、Δ1910hk/rr和CΔ1910hk/rr体外生物学特性比较研究。结果表明：Δ1910hk/rr的生长速率要低于WT和CΔ1910hk/rr。透射电镜结果显示,Δ1910hk/rr形态结构未发生改变。定性和定量的溶血性试验结果表明,与WT相比,Δ1910hk/rr的细菌溶血活性并没有发生显著变化。中性粒细胞(PMN)介导的杀伤实验结果显示,双组分调控系统1910HK/RR缺失后使得SS2抵抗中性粒细胞(PMN)的杀伤能力降低。体外细胞粘附和侵染试验结果表明,Δ1910hk/rr对HEp-2细胞的粘附和侵染能力降低,但是粘附能力下降更为显著。3.双组分1910HK/RR缺失后对SS2致病性的影响为了进一步评价双组分调控系统1910HK/RR对SS2致病性的影响,在CD-1小鼠和断奶仔猪身上开展了动物实验。分别用菌株WT、Δ1910hk/rr和CΔ1910hk/rr感染CD-1小鼠。半数致死量(LD5o)结果为,Δ1910hk/rr为1.22×109CFU,而WT为4.14×107CFU野毒菌株,突变株是野生菌株的29.5倍,Δ1910hk/rr的毒力与WT相比下降非常显著。发病率和存活率试验：参考小鼠半数致死量的结果,采用了108CFU的剂量来进行试验。分别用Δ1910hk/rr、CΔ1910hk/rr和WT感染小鼠,观察12 d。其结果为：与WT相比,Δ1910hk/rr发病率降低,死亡率为10%,感染后小鼠脑和肺组织无明显的病理损伤。WT与CΔ1910hk/rr的死亡率分别为70%和80%。组织器官载菌量结果显示,Δ1910hk/rr感染组小鼠的心、肝、脾、肺、肾、脑、血等组织活菌数都要低于WT。进一步以105CFU剂量感染5周龄左右断奶仔猪。结果为：感染后48 h内WT组死亡率为66.7%(4/6)；存活的2头仔猪也出现典型的症状,而Δ1910hk/rr组观察到21d,死亡率为16.7%(1/6)：心、肝、脾、肺、肾、脑、血液、关节等组织载菌量比较分析发现,Δ1910hk/rr组也低于WT组。以上结果说明,双组分1910HK/RR缺失后SS2的致病性明显降低。4.双组分调控系统1910HK/RR调控SS2致病性机制的初步研究为了探讨双组分调控系统1910HK/RR调控SS2致病性的可能分子机制,首先,通过基因表达谱芯片片,开展了Δ1910hk/rr与WT差异表达基因分析。结果显示：在整个基因组2194个基因中,Δ1910hk/rr有219个基因转录水平发生了变化,占整个SS2基因组9.98%。其中105个基因下调表达1.5倍以上,114个基因上调1.5倍以上。有50个基因表达下调2倍以上,32个基因表达上调2倍以上。选取与氨基酸转运、代谢、毒力、荚膜合成等相关的24下调表达的基因进行荧光定量PCR验证,其结果也全部下调,证实了芯片结果的可信性。为了研究1910HK/RR与唾液酸合成相关的4个基因(NeuB.NeuC.NeuD.NeuA)的结合特性,克隆、表达并纯化双组分调控系统1910HK/RR的反应调控蛋白1910RR,经EMSA证实,唾液酸合成相关基因启动子能与1910RR结合,初步证实了唾液酸合成相关基因受1910HK/RR的调控。更多还原

【Abstract】 Streptococcus suis serotype 2 (SS2) is one of the most important swine pathogens, and an emerging, life-threatening zoonotic agent in both pigs and humans. Based on the CPS antigens of S. suis,35 serotypes have been characterized. SS2 is most commonly and most virulent of these serotypes. SS2 cause great economic loss to the pig industry worldwide. Due to high economic losses as well as threat to human life, the study of SS2 pathogenesis, and the development of new vaccines is crucial for prevention and control of this disease.Development of the disease requires temporal and coordinated expression of a series of genes that allow the prospective pathogen to shift to its pathogenic state and adapt to a hostile environment in the host. TCS control the expression of virulence factors in a wide range of bacterial species in response to external stimuli. For bacterial pathogens, they have evolved virulence factors and regulation systems that allow adaptation to different environments within a host ensuring their survival. TCS generally consist of a sensor kinase and response regulator and are the major means by which bacteria recognize and respond to a variety of environmental stimuli. Many results have implicated these systems as playing an important role in the regulation of a variety of essential processes including cell-cycle progression, pathogenicity, and development.Two SS2 strains 05ZYH33 and 98HAH12 isolated in China have been sequenced. The results predict that there are 15 TCS in both the strains together with single, unpaired response regulator. To date, only the SalK/SalR system and two orphan response regulators, RevS and CovR.In this work, we constructed a mutant strain of the TCS 1910HK/RR, designatedΔ1910hk/rr and measured its virulence in vitro and in vivo. So the following researches were explored.1. Construction and verification of SS2 mutant strain (Δ1910hk/rr) and complementation strain (CΔ1910hk/rr)The upstream and downstream flanking regions of 1910hk/rr were separately amplified fromSS 2 wild-type strain SC19 genomic (WT) DNA respectively. Followed by digestion with the corresponding restriction enzymes, the PCR products were directly cloned into a pSET4s vector. Then, the plasmid, pSET4s-1910hk/rr was electroporated into WT, and the resultant strains were grown at 28℃in the presence of spectinomycin (100μg/mL) selection and subsequently passaged at 37℃in the absence of spectinomycin selection as described previously. Successful deletion of the 1910hk/rr was confirmed by PCR and RT-PCR. For complementation analysis, a DNA fragment containing 1910hk/rr and its upstream promoter were amplified and cloned into the E.coli-S.suis shuttle vector pAT18. Then the recombinant plasmids were electrotransformed into the mutants to screen the complemented strains CΔ1910hk/rr.2. Biology characteristic research of the SS2 mutant strainΔ1910hk/rr, complementation strain CΔ1910hk/rr and SS2 wild type srain (WT)The results of the growth curve ofΔ1910hk/rr、CΔ1910hk/rr and WT showed thatΔ1910hk/rr grow slower than that of WT. The hemolytic activity of wild-type strain was more high higher than that of mutant strain. However, there were not significant difference-betweenΔ1910hk/rr and WT. The hemolytic activity of complementation strain was restored, and was about two times higher thanΔ1910hk/rr. But it did not reach the level of WT. To assess whether the lack of 1910HK/RR affected the cellular adhesion of SS2, the adherence efficiencies of the WT and theΔ1910hk/rr mutant to human continuous laryngeal epithelial cell line Hep-2 cells were compared. The results showed that WT showed significantly more adhesion to both Hep-2 cells than theΔ1910hk/rr, indicating the role of 1910HK/RR as an important mediator in the cellular-adhesion process. LD50 values were 4.14×107CFU for WT,1.22×109CFU forΔ1910hk/rr. Compared with parent strain,Δ1910hk/rr was attenuated 29.5 fold. The effect of 1910HK/RR on PMN killing was further examined. PMN were incubated with two strains for two hour and the bactericidal activity of PMN cells was determined. Remained bacteria were decreased in theΔ1910hk/rr group. In contrast, the WT strain still multiply when incubated with PMN, indicating that 1910HK/RR is able to enhance the bactericidal activity of PMN.3. Impact of the 1910HK/RR on the virulence of SS2 WTIn order to evaluate whether the 1910HK/RR contributes to the virulence of SS2 in vivo, an experimental infection of CD-1 mice was performed. Morbidity and mortality analysis of CD-1 mice was performed to study pathogenesis of 1910HK/RR. Five-week-old CD-1 mice were intraperitoneally inoculated with 108 CFU/mL bacteria and the survival was monitored over a 12-day period. The results showed that almost all animals in the WT group presented severe clinical signs of sepsis, such as depression, rough hair coat, swollen eyes, weakness and prostration during the first three days post-infection. Seven mice died from septicemia in the WT group during 48 hour post-inoculation. In contrast, only one mice died in theΔ1910hk/rr group during the 48 hours post-inoculation and all of remaining nine mice did not present severe clinical signs of sepsis associated with SS2 infection during the trial, recovering after 4 days post-inoculation. In order to further confirm the clinical signs ofΔ1910hk/rr mutant in vivo, bacteria loads of organs were monitored. According to results above, mice were intraperitoneally inoculated with either the strainΔ1910hk/rr or WT at a dose of 1×107 CFU. Results showed that bacteria loads in blood following inoculation were lower in theΔ1910hk/rr group, suggesting that theΔ1910hk/rr could not multiply effectively in organs compared with WT.The contribution of 1910HK/RR to SS2 virulence was assessed by infecting piglets with theΔ1910hk/rr mutant or WT strain. All six piglets inoculated intranasally with WT strain at the dose of 1×105 CFU developed hyperthermia and depression within 48 hours, including two of which that died withing 24 hours. Later, most of the typical disease symptoms, including limping, swollen joints, shivering, central nervous system failure and respiratory failure were observed. Four of six piglets (4/6) infected with WT strain died within 48 h post-infection. In contrast, none of the piglets infected with theΔ1910hk/rr developed any of the clinical signs mentioned above within the first 24 hours with exception of one which was slightly depressed. However, one piglet died within 48 hours. The remaining five recovered from fever showing none of other typical disease symptoms and remained healthy throughout the 21 day experiment. To better evaluate the virulence attenuation ofΔ1910hk/rr, colonization experiments and histopathological examinations were carried out. The results showed that almost all the bacterial counts recovered from different tissues ofΔ1910hk/rr mutant infected-piglets were significantly lowercompared with WT infected-piglets. Notably, the bacterial counts from theΔ1910hk/rr infected group was lower than WT. Histopathological examination of brain and lung tissues demonstrated that the blood vessels of brain from the WT infected group were filled with numerous macrophages and neutrophils compared to theΔ1910hk/rr infected group. In fact, the presence of high numbers of leukocytes suggests that the exacerbated inflammatory response might be the cause of death within the first 48 hours.4. Role of 1910HK/RR in the virulence regulation of WTTo gain further insights into the network mediated by 1910HK/RR, DNA microassay, quantitative PCR was applied to reveal the differential transcription profiles between the strain WT with theΔ1910hk/rr independently. In this study we used a microarry which was made by using available the SS2 05ZYH33 strain whole genome sequence. To compare with the wild type SS2 strain SC19 gene expression,Δ1910hk/rr shows that 105 genes were down-regulated 1.5-fold and 114 genes were upregulated 1.5-fold. In total, the absence of 1910hk/rr led to changed expression of over 200 genes. Of the 114 upregulated genes,24 genes were investigated. They can be roughly categorized into the following four groups:(Ⅰ) Genes involved in ABC-type amino acid transport and metabolism; (Ⅱ) Genes involved in metabolism; (Ⅲ) Genes encoding membrane proteins and some proteins of unknown function, associted with virulence. (Ⅳ) Genes encoding sialic acid synthesis gene. The intereaction of DNA promoter with regulator 1910RR was confirmed by EMSA.更多还原