【作者】 陈博；

【导师】 金梅林；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2012， 博士

【摘要】 猪链球菌(Streptococcus suis, S. suis)是一种重要的细菌性猪病病原,其常给全球的养猪业造成巨大损失。当感染S. suis后,病猪通常会出现脑膜炎,关节炎,心内膜炎,以及败血症等症状。该菌按照血清型的不同,可分为33个血清型(1型-31型,33型和1/2型),其中猪链球菌血清型2型(Streptococcus suis serotype2, SS2)在世界范围内被广泛报道为最主要的致病性血清型。另外,S. suis也是一种重要的人畜共患病病原。从丹麦的第一例人感染猪链球菌病例开始,S. suis已经在全球至少20个国家造成超过700人感染。尽管猪链球菌已在全球引起了高度的关注,然而当前对其致病机理的研究还很有限。病原菌粘附到宿主细胞或组织表面以及其随后的侵入与扩散过程是病原菌致病的关键步骤。并且病原菌与宿主之间的相互作用参与着所有这些过程。因此,研究病原菌的致病机理也就是研究病原菌与宿主的相互作用过程。本研究试图以一些S. suis的表面毒力相关蛋白为诱饵,通过鉴定该蛋白与宿主细胞之间的相互作用,为解释其生物学功能与致病机理提供理论依据；另一方面,能够与宿主细胞发生相互作用的细菌表面蛋白大多参与着细菌的致病过程。因此,本研究希望能够通过大规模系统的方法将这部分蛋白鉴定出来,其将为进一步的研究病原菌的致病机理提供研究基础；病原菌的毒力相关蛋白通常也是其重要的保护性抗原,因此本研究所鉴定的细菌表面蛋白极有可能被开发成为疫苗候选的分子,为解决猪链球菌病得防控提供帮助,而本研究也尝试去评价这部分蛋白的保护效力。现将主要研究结果报告如下：1.猪链球菌保护性抗原HP0197与宿主细胞之间的相互作用的研究在本研究前期的工作当中,HP0197蛋白已被鉴定为SS2新型的保护性表面抗原和毒力相关蛋白。然而该蛋白的生物学功能尚不清楚,其在致病过程中的作用还有待于进一步证实。在本研究中,我们试图通过鉴定HP0197与宿主细胞的相互作用过程,进而为阐明该蛋白的生物学功能提供理论依据。首先,我们通过间接免疫荧光实验证实了HP0197能够与Hep-2细胞发生相互作用。进一步实验显示,这种相互作用可以被肝素和胰酶完全阻断,其暗示着宿主细胞表面的类肝素的分子是HP0197的细胞受体。另外,HP0197还被发现能够与葡萄糖相结合,因此HP0197可能参与着Ssuis的能量代谢。为了证实这个推测,我们比较了△hp0197菌株和亲本株在不同培养时期的HPr蛋白的生物学活性。结果显示：当hp0197基因缺失后,菌株体内的HPr蛋白的磷酸化随之改变,因此能够进而影响碳代谢调节基因CcpA的功能。2.鉴定猪链球菌2型表面蛋白与宿主细胞表面分子之间的相互作用病原菌与宿主蛋白质之间的相互作用参与着病原菌所有的感染过程。研究病原菌与宿主之间的相互作用能够我们全面理解细菌病的致病机理。尽管目前这个领域已有少量的研究,但仍有待于全面的系统鉴定病原菌与宿主之间的蛋白质相互作用过程。在本研究中,我们建立了一种名为基于亲和层析的表面蛋白质组学(Affinity Chromatography-based Surface Proteomics; ACSP)的新研究方法,该方法将亲和层析技术和shotgun蛋白质组学技术(LC-MS/MS)相结合,可以用来大尺度系统的研究病原与宿主之间的相互作用。为了完成ACSP实验,我们以锚定了天然的Hep-2细胞表面分子的树脂为诱饵,用来从S. suis表面蛋白中捕获潜在的互作分子。随后,我们利用LC-MS/MS技术从捕获物中鉴定出40个潜在的表面互作蛋白,这些蛋白中有3个蛋白已在前期的研究中被证实。我们选取了8个重要的表面互作蛋白,并证实了其与Hep-2细胞的粘附作用。另外,3个新鉴定的表面互作蛋白的重组蛋白以及多克隆抗体能够显著的抑制S. suis与Hep-2细胞的粘附过程,因此这3个蛋白极有可能在Ssuis与Hep-2细胞的相互作用过程中发挥着重要的作用。通过使用这个例子,我们证实了ACSP技术是一个非常有价值的研究病原菌与宿主相互作用的实验工具。3.猪链球菌免疫原性蛋白HP0272的保护力评价S. suis是一个重要的猪细菌病病原,其同时也是一个人畜共患病病原。开发新的有效疫苗能够帮助我们控制S. suis的感染。HP0272是一个细菌表面免疫原性表面蛋白,其保护效力有待进一步的评价。在本研究的小鼠模型当中,纯化的重组HP0272能够诱导显著的抗体水平,并保护小鼠以抵抗致死剂量的SS2人工感染。另外,实时荧光定量PCR也证实HP0272为体内诱导抗原。该基因在一半的S. suis参考血清型菌株以及大多数SS21临床分离菌株的基因组中均存在。所有的这些结果暗示着HP0272是一个潜在的新疫苗有效成分。更多还原

【Abstract】 Streptococcus suis (S. suis) is an important swine pathogen which causes an economical problem in the swine industry. The manifestations of S. suis infection in swine are meningitis, arthritis, endocarditis and septicemia. Thirty-three serotypes (types1-31,33and1/2) have been described on the basis of the capsular polysaccharides, and S. suis serotype2(SS2) is most commonly associated with diseases in pigs in worldwide. Moreover, it’s also the causative agent of serious infections in humans. From the first infection case reported in Demark, more than700infection cases were reported at least20countries all over the world. Although S. suis infection has attracted great attention from the scientific community and the popular press, our current understanding of the S. suis pathogenesis remains limited. The attachment of bacteria to host cells and tissues and their subsequent invasion and spreading are key processes during pathogenesis. Interactions between the bacteria and host cells are responsible for these processes. In this study, we try to provide the theoretical basis to explain the pathogenesis of S. suis by identifying the surface protein-protein interactions between the S. suis and host cells. In the other hand, the surface proteins of pathogenic bacteria which could interact with the host cells play important roles in the pathogenesis of bacteria. Therefore, in the present study, we also want to identify more surface interacting proteins (SIPs) of S. suis by an interactomics method. And the identified proteins could be applied as the candidate proteins to further indentify their pathogenic functions in infection processes. Based on the results, we would screen the SIPs to develop the novel vaccine. And it would be benefit to infection control. The principal results were described as follows:1. The interactions between the protective antigen HP0197and host cellsIn previous works, the protective antigen HP0197had been identified as a virulence-involved protein. However, the biological functions of HP0197remain unknown, and its roles in infection processes need to be further identified. In the present study, we try to indentify the interactions between the HP0197and host cells. And it would help us to define the biological functions of HP0197. First, we confirmed that HP0197could adhere to the surface of Hep-2cells by indirect immunofluorescence assays. The further studies indicate that the interactions between HP0197and Hep-2cells could be inhibited completely by heparin and trypsin. Therefore, it suggests that the heparin-like saccharide would be the receptor of HP0197on the host cells. In addition, the interaction between HP0197and glucose was also confirmed, suggesting that HP0197may involve in the carbon metabolism of S. suis. To proof the supposition, we compare the activity of HPr in Ahp0197strain and parent strain. The results indicate that the phosphorylation of HPr was changed when the hp0197gene was deleted, and it would further affect the biological activity of CcpA.2. Identification of the interactions between the surface proteins of S. suis and the surface molecules of host cellsProtein-protein interactions between bacteria and their hosts are responsible for all types of infection processes. The investigation of the bacteria-host crosstalk can provide a comprehensive understanding of the pathogenesis of bacterial disease. Despite scattered efforts in this field, a systematic identification of interactions between host and bacterial proteins remains unavailable. Here, we develop ACSP (Affinity Chromatography-based Surface Proteomics), which combines affinity chromatography and shotgun proteomics (LC-MS/MS), to investigate the interactions on a large-scale. Using ACSP, the potential surface interacting proteins (SIPs) of SS2were captured by the chromatographic resin which was immobilized with the native surface molecules of Hep-2cells. And then40potential SIPs were identified from the preys by LC-MS/MS, including3SIPs that have been previously reported in the literature. We selected8important SIPs and confirmed their ability to adhere to Hep-2cells. Additionally,3newly identified SIPs, or their polyclonal antibodies, were found to significantly inhibit the adherence of SS2to Hep-2cells, indicating their essential role in the interaction between SS2and Hep-2cells. Using this example, we show that ACSP represents a new valuable tool for investigating the bacteria-host interactions.3. Evaluation of the protective efficacy of a novelly identified immunogenic protein, HP0272, of Streptococcus suisSS2infection is a major cause of sudden death in pigs and is of concern for humans as it has strong zoonotic capabilities. Developing novel effective vaccines would be beneficial to control SS2infection. HP0272is a novel immunogenic surface protein; its protective efficacy remains to be evaluated. The present mouse model found that the purified recombinant HP0272could elicit a significant humoral antibody response, and to confer complete protection against a lethal dose of SS2infection. In addition, real-time PCR confirmed that in vivo-induced antigen existed in most SS2field pathogenic strains, and in half of all reference strains of different serotypes of S. suis. The results indicate that HP0272has the potential as an effective component of a new vaccine.更多还原