【作者】 王雅；

【导师】 金梅林；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2011， 硕士

【摘要】 猪链球菌(Streptococcus suis,S.suis)是当今危害养猪业的一种重要病原菌,可导致猪败血症、脑膜炎、肺炎、心内膜炎、关节炎等,在养猪业造成了很大的危害和损失。依据细菌表面的荚膜多糖(CPS)抗原差异,可以将S.suis分为33种荚膜血清型,即1-31、33和1/2型。其中猪链球菌2型(SS2)是临床分离株中致病力最强,同时也是目前分离率最高的血清型。该型菌可引起人的感染甚至死亡,是一种重要的人畜共患病。1998年我国江苏省部分地区猪链球菌2型感染导致25人感染,14人死亡。2005年我国四川省部分地区爆发的猪链球菌2型病导致204人感染,38人死亡。猪链球菌对人类健康带来了极大的威胁。目前对猪链球菌的致病机制进行了很多研究,但其机制并不完全清楚。对猪链球菌毒力(相关)基因的研究有助于阐述其致病机制。荚膜多糖(CPS)是猪链球菌2型重要的毒力因子,然而许多无毒菌株也含有荚膜多糖,这表明其它的毒力(相关)因子在猪链球菌致病过程中也起着十分重要的作用。猪链球菌的某些分泌性蛋白可能在其致病过程中起到了至关重要的作用,这些分泌性蛋白只要达到一定的浓度,并不需要细菌与宿主细胞接触就能发挥作用。其中最重要的分泌性蛋白就是溶血素(SLY),SLY被证实除了具有细胞毒性外,还能阻断补体介导的吞噬和杀伤作用。SLY在猪链球菌2型侵入和裂解细胞的过程中以及导致脑膜炎过程中发挥重要作用。同时溶血素阳性的菌株通常更容易导致系统性感染。基于SLY在猪链球菌的致病过程中的作用,一些学者认为SLY可能是欧洲和亚洲猪链球菌最为重要的毒力相关因子。有研究指出,在许多2型和9型毒力菌株中,存在一个开放阅读框,编码型Ⅲ溶血素,但目前并没有实验证据表明除了SLY,猪链球菌还能表达其他的活性溶血素。因此本研究对猪链球菌2型的Ⅲ型溶血素基因(slyrp)的功能进行了研究,探讨其在SS2裂解红细胞及致病过程中的作用。本研究通过同源重组基因敲除法成功构建了2005年中国四川资阳分离强毒株SS205ZY的slyrp基因缺失菌株(△slyrp)。作为对照,同时构建了sly基因缺失菌株(△sly)及双基因缺失菌株(△sly△slyrp)。并比较了野生菌株和突变菌株的溶血能力以及对小鼠的致病力。发现sly基因敲除后,SS2裂解红细胞的能力下降明显,slyrp基因敲除后可导致SS2裂解红细胞的能力有轻微下降,而双基因缺失突变菌株△sly/△slyrp的溶血能力完全丧失。sly基因敲除后,猪链球菌对小鼠的致病力也有一定程度的减弱,但仍表现较高的致病力,slyrp基因敲除后,猪链球菌对小鼠的致病力没有变化,而双基因缺失菌株的致病力显著下降。结果表明：在猪链球菌2型裂解红细胞的过程中,slyrp只裂解少量红细胞,sly起主要裂解作用,slyrp起协同作用；slyrp在SS2感染过程中起到一定的作用,但对其毒力强弱不起决定作用；sly是SS2裂解细胞的主要的毒力因子,但很显然不是造成SS2高致病力的唯一的毒力因子。更多还原

【Abstract】 Streptococcus suis is an invasive porcine pathogen associated with septicemia, meningitis, pneumonia, endocarditis, arthritis and other diseases. The pathogen constitutes a major health problem and great loss in the swine industry worldwide. Thirty-three serotypes (types 1-31,33 and 1/2) have been described based on capsular polysaccharides, and S. suis serotype 2 (SS2) is considered to be the most pathogenic as well as the most prevalent capsular type in diseased pigs. SS2 might also cause diseases even death in humans. Recently, two large-scale outbreaks of human SS2 in China (25 cases with 14 deaths in Jiangsu in 1998, and 204 cases with 38 deaths in Sichuan in 2005) showed that Streptococcus suis is actually a great threat to human health. Although a set of virulence traits have been identified, molecular mechanisms of invasion and damage of host tissues by the bacteria are still far from clear. Studies on virulence-associated factor contribute to explain the pathogenicity of S. suis.It has been shown that the polysaccharide capsule is a great recognition virulence factor of S.suis. However, some avirulent strains seem to be fully encapsulated. This suggests that some other virulence factors may play important role on virulence of S.suis. Some Secreted proteins may have important functions in pathogenesis of S.suis. These secreted proteins can damage host cells depending on diffusion in tissue in order to reach critical concentration but do not require bacteria-host cell contact. SLY has been believed to be one of the most important secreted proteins. In addition to its cytotoxic activity, SLY plays critical role on the process of S.suis infecting host cell by blocking the complement-mediated phagocytosis and killing role. SLY contribute to Streptococcus suis type 2 (SS2) invasion and cell lysis and lead to meningitis. Furthermore, s/y-positive strains are usually more prone to lead to systemic infection. Due to these functions, some scholars believe that the SLY may be the most important virulence associated factors of Europe and Asia strains.It has been pointed out that an ORF in many virulent serotype 2 and 9 strains encodes a putative hemolysin type III. However, at present there is no experimental evidence for expression of any other active hemolysin except suilysin. Therefore this expriment was conducted to study the function of hemolysin type III gene(slyrp) of Streptococcus suis type 2 to investigate the role of slyrp in cleavage of red blood cells and pathogenicity of SS2. The SS205ZY isogenic suilysin mutant Aslyrp was constructed through homologous recombination, as control, Aslyrp and the two genes knock out mutant Asly/slyrp were construct. The hemolysis capability and virulence to mice between wild type strain and the gene deleted mutants were compared. The results indicated that the isogenic sly mutant showed significant decreased hemolysis capability; the isogenic slyrp mutant showed slight decreased hemolysis capability and the the two genes knock out mutant Asly/slyrp showed no hemolysis capability. The sly gene knock out mutant showed relative lower virulent to mice; slyrp gene knock out mutant showed the same virulent to mice as wild type strain; the two genes knock out mutant Asly/slyrp showed considerable decreased virulent to mice.The results indicated that:sly plays an important role in invading and lysis of host cells by SS2, and slyrp does synergistic effect to sly. Slyrp plays relative role but not decisive role in SS2 infection. Sly is the main virulence factor, but apparently is not the only virulence factor that leads to the high pathogenicity of SS2.更多还原