【作者】 张萍；

【导师】 范红结；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2016， 硕士

【摘要】 猪链球菌(Streptococcus suis,S.suis)是一种重要的人畜共患病病原,可引起猪和人的广泛性感染,表现为脑膜炎、败血症、关节炎及心内膜炎等。在33个血清型中,猪链球菌2型(Streptococcus suis type2,SS2)流行最广,致病性最强,危害最重,给养殖业造成了巨大的经济损失,并危害公共卫生安全。尽管已报道了该菌的多种毒力因子,但其致病机理仍不清晰。本研究通过转座子突变技术筛选SS2抗吞噬相关基因,并对筛选所得hsdS基因的功能进行验证。研究结果有助于进一步阐明猪链球菌2型抗吞噬机理。1、猪链球菌2型抗吞噬相关基因的筛选抗吞噬是SS2抵抗机体先天性免疫,逃避免疫杀伤,引发猪链球菌病的一项重要功能。为发掘SS2抗吞噬相关基因,进一步研究SS2逃避宿主先天性免疫机制,本实验利用含转座子TnYLB-1的pMar4S质粒构建ZY05719菌株突变体文库,通过BV-2细胞吞噬试验获取抗吞噬能力减弱突变株,从而分离、鉴定SS2抗吞噬相关基因。试验成功构建了含2000株突变株的突变体文库,筛选出4株抗吞噬能力减弱的突变株(P<0.05)。该4株突变株的吞噬率分别高于野生株:1.976、2.29、1.16和1.63倍,转座子插入基因分别为:通透酶基因(ZY05719_RS02540),Ⅰ限制性修饰系统的S亚基基因(ZY05719_RS06850),1,4-二羟基-2-萘八异戊烯转移酶基因(ZY05719_RS08285)和支链氨基酸转运蛋白相关基因(ZY05719_RS03750)。研究结果为进一步研究SS2抗吞噬机制提供了基础。2、猪链球菌2型hsdS基因抗吞噬功能的验证hsdS是猪链球菌2型Ⅰ型R-M系统基因簇中的一个基因,编码蛋白HsdS,形成限制性内切酶和甲基转移酶。本章通过构建穿梭载体pSET2::hsdS导入至转座子突变株MhsdS,从而构建了突变株的互补株CMhsdS,验证hsdS在SS2抵抗小鼠小胶质细胞BV-2吞噬过程中的作用。试验结果显示互补株CMhsdS与突变株MhsdS相比,抗吞噬能力恢复了 45.4%,且差异显著(P<0.001)。此外,系列实验显示MhsdS在小胶质细胞内存活,全血存活,酸、氧耐受,刺激小胶质细胞产生细胞因子及一氧化氮等方面的能力与野生株相比存在显著差异(P<0.05);而互补株CMhsdS与突变株相比,除全血存活和酸性耐受能力没有得到恢复,以上其他方面的能力均得到一定的恢复,且与突变株存在显著差异(P<0.05)。转录组结果显示突变株MhsdS与野生株ZY05719有56个差异表达基因,其中33个基因上调表达,23个基因下调表达,主要涉及过程包括能力代谢,细菌细胞壁和膜功能等。综上所述,hsdS基因能够促进SS2抵抗小胶质细胞的吞噬作用,同时在SS2致病过程中具有多种生物学功能。3、猪链球菌2型Ⅰ型R-M系统抗吞噬功能的分析本试验所研究的SS2Ⅰ型R-M系统基因簇包含hsdR,hsdM,hsdS和hsdS’四个基因,其中hsdS是突变株MhsdS中转座子TnYLB-1的插入基因,与SS2抵抗小胶质细胞吞噬功能有关。为进一步研究Ⅰ型R-M系统对SS2抵抗BV-2细胞吞噬功能的作用,本试验利用温敏型穿梭质粒pSET4s分别成功构建了hs R和hsdM的缺失株(△hsdM,△hsdR),hsdS的部分和全长缺失株(△hsdSp,△hsdSf),以及hsdS’的部分和全长缺失株(△hsdS’p,△hsdS’f)。与野生株相比,各缺失株生长速率无明显差异,缺失株△hsdSf、△hs和△hsdR抵抗小胶质细胞吞噬能力显著降低(P<0.05)。各缺失株诱导小胶质细胞产生TNF-α的量均高于野生株,但仅缺失株△hsdSp和△hsdSf差异显著(P<0.05);而诱导产生MCP-1的量则基本低于野生株,除缺失株△hsdS’f和△hsdR外,其他差异均显著(P<0.05)。因此,hsdS的抗吞噬相关功能可能与整个Ⅰ型R-M系统的生物学功能相关。更多还原

【Abstract】 Streptococcus suis(S.suis)is considered an important zoonotic agent responsible for a wide variety of diseases in pigs and human,including meningitis,septicemia,arthritis and endocarditis.S.suis serotype 2(S.suis 2,SS2)is one of the most virulent,hazardous and frequently isolated serotype among 33 serotypes,causing important economic losses to porcine industry and endangering public health security.Pathogenic mechanism of SS2 is too complex to explain despite numorous of virulence factors had been reported.In this study,we identified genes involed in anti-phagocytosis by using technology of transposon mutagenesis.Biological function of gene hsdS was analyzed by multiple experiments.This study will lead to a better understanding of the anti-phagocytosis mechanism of SS2.1.Selection of anti-phagocytosis-related genes in Streptococcus suis serotype 2Anti-phagocytosis is an essential feature of SS2 to escape host innate immune system and cause Streptococcus suis diease.This research was aimed at identifying genes involed in features of anti-phagocytosis by constructing SS2 mutant library and phagocytosis test.Finally,we successfully constructed a mutant library containing 2000 mutant strains and selected 4 mutants with high phagocytic rate which means weakened ability in anti-phagocytosis.Genes inserted by transposon in the 4 mutants are as follows:ZY05719_RS02540,ZY05719_RS06850,ZY05719_RS08285 and ZY05719_RS03750,with product of permease,type I restriction endonuclease subunit S,1,4-dihydroxy-2-naphthoate octaprenyltransferase and branched-chain amino acid ABC transporter,respectively.This study will provide a foundation to the research of anti-phagocytosis mechanism of SS2.2.Verification of anti-phagocytic function of hsdS in Streptococcus suis serotype 2hsdS is a gene in type I R-M system gene cluster of SS2,encoding protein HsdS,forming retriction endonuclease and methyltransferase.In this study,we verified the anti-phagocytic function of hsdS by constructing complementation strain CMhsdS using a shuttle vector,pSET2.Results of phagocytosis test showed that the anti-phagocytic ability of complementation strain CMhsdS was recovered for 45.4%compared to mutant strain MhsdS(P<0.001).In addition,compared to wild strain ZY05719,mutant strain MhsdS was significantly different in vability in microglia,pig whole blood,acid condition of pH5 and oxygen stress with 1mM H2O2,and ability in stimulate production of cytokines and nitric oxide by microglia(P<0.05).Biological characteristics of complementation strain CMhsdS were partly recovered.Transcriptome analysis showed that there were 56 genes expressed differently between wild strain and mutant strain,among which 33 genes were up-expressed while 23 were down-expressed.In summary,hsdS is indeed a gene related to function of anti-phagocytosis,and also a gene contributed to many biological characteristics in SS2.3.Analysis of anti-phagocytic function of type Ⅰ restriction-modification system in Streptococcus suis serotype 2Type Ⅰ R-M system studied in our research contains four genes:hsdR,hsdM,hsdS and hsdS’.hsdS is the inserted gene by transposon TnYLB-1,verified to be anti-phagocytic related.In order to study the function of type I R-M system in anti-phagocytosis,six defective mutant strains(△hsdM,△hsdR,AhsdSp,△hsdSf,△hsdS’p and △hsdS’f)were constructed by temperature-sensitive shuttle vector pSET4s.Compared with wild strain ZY05719,mutant strains had no significant difference in growth rate,but were partly different in phagocytic rate and ability in stimulate production of cytokines by microglia.Therefore,the anti-phagocytic function of hsdS may related to the biological functions of type Ⅰ R-M system.更多还原