猪链球菌2型fbps基因突变株的构建及其生物学特性的研究
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摘要
猪链球菌(Streptococcus suis, S. suis)是导致猪链球菌病主要的病原菌。该菌可分为33个血清型,其中猪链球菌2型(Streptococcus suis type2, SS2)致病性强、流行最广泛并且在临床上,该型是分离率最高的血清型之一。SS2可引起猪的脑膜炎、化脓性肺炎和心肌炎等疾病。SS2可通过直接感染特定从业相关人群,严重的病例发生中毒性休克综合症,导致多脏器衰竭及死亡,是一种重要的人畜共患病。近几年猪链球菌病的几次爆发流行,因此SS2成为了国内外广泛关注和研究的热点,尤其是关于其致病和免疫防治。
SS2对宿主的粘附过程可能是其致病过程中一个关键的步骤。SS2和宿主细胞的粘附并非一个简单的过程,在细菌和细胞表面都有多种相关因子参与了此过程,因此研究在此过程中发挥作用的关键因子可能会对SS2致病机理阐释发挥重要作用。
猪链球菌2型基因组中的fbps基因编码了一种具有纤连蛋白结合位点的表面膜蛋白(fibronectin binding proteins, FBPS),和它具有同源性的同样具有Fn结合活性的蛋白在其他链球菌的感染中发挥着关键的作用,因此推断,FBPS可能是猪链球菌感染的吸附过程中的一个重要的参与蛋白。
在此背景之下,我们以SS2高致病性菌株SC19作为亲本菌株,以纤连蛋白结合蛋白编码基因作为研究对象,构建了fbps基因的缺失突变株,并在此基础上分析fbps基因的缺失对SS2基本生物学性状以及对致病力的影响,主要研究内容包括：
1温敏型自杀性重组质粒的构建
以2005年四川分离的SS2高致病性菌株SC19为亲本菌株,分别扩增fbps基因的780bp上游序列和81bp的下游序列,并利用融合PCR的方法获得上下游同源臂全长片段并连接到pMDx-18T载体,构建克隆载体pMDx-18T-f,分别用EcoR1和BamH1同时酶切pMDx-18T-f和pSET4s载体后连接,构建自杀性重组转移质粒pSET4s-fbps。
2纤连蛋白结合蛋白fbps基因突变株的构建
将重组质粒pSET4s-fbps电转化亲本菌株SC19感受态细胞,以壮观霉素抗性标记和温度敏感双重筛选,得到fbps基因缺失突变菌株。并进一步通过PCR等方式对筛选出的突变菌株进行验证。
3纤连蛋白结合蛋白fbps基因突变株生物学性状的研究
在构建的fbps基因突变株的基础上,研究缺失菌株的遗传稳定性,体外生长和增殖的能力等生物学性状。结果表明,连续传代十代突变株均能稳定遗传。野生株和突变株的生长没有显著的差异,fbps基因的缺失不会影响SS2的生长和繁殖的速度。
4fbps对宿主的吸附侵入以及毒性作用
以Hep-2细胞为模型,对比研究fbps缺失菌株和SC19野生型菌株粘附能力和侵入能力,以培养液中LDH的渗出为参照,测定培养液中LDH含量,比较突变株和野生菌株对细胞毒性作用。结果表明,fbps基因缺失突变株对Hep-2细胞的粘附能力和亲本株SC19相比下降了78%；对细胞侵入能力下降23%；测定LDH的浓度发现fbps基因突变株比野生型的毒性降低了32%。
5突变株致病性研究
将野生型菌株SC19和fbps基因缺失突变株分别以107cfu/ml,108cfu/ml和109cfu/ml的剂量腹腔注射昆明鼠(1m1)。结果显示,注射突变株的死亡率远远低于注射野生型SC19菌株的试验组。不同时间点剖杀小鼠不同器官的细菌动态分布表明,缺失了fbps基因导致细菌的组织定植能力显著下降。病理组织学观察发现SS2fbps基因缺失突变株和野生型在小鼠中病变范围类似,主要引起肺部,脑部和心脏的病变,但是突变株引起的病变程度较轻微,说明fbps基因缺失突变株致病性显著降低。
Streptococcus suis is an important pathogen of swine, and33serotypes have been identified based on their capsular antigen, of which the Streptococcus suis serotype2is the most important and frequently isolated from the clinical samples. SS2can cause a lot of serious diseases, such as meningitis, septicemia, arthritis, endocarditis, pnuemonia and even acute death in swine. SS2can also infect humans who were associated with pigs or swine industry directly; the serious cases would have STSS which leads to multiple organ failure or even death. It is an important zoonosis pathogen and is paid worldwide attention. Recent years several outbreaks of SS2have seriously harmed the swine industry and human health, then it was paid great attention by researchers of China or other countries.
The adhesion of SS2to the host maybe a key step in the pathopoiesis processes, so many works about adhesins have been carried out. But the adhesion of SS2to the host cells is not a simple way, many different components of SS2or the host cells were involved in it, so to find out which one play the major role in this process is usefully and necessarily to furter explain how SS2do harm to the hosts.
In the SS2genome there is a gene fbps encoding a surface membrane protein FBPS, which has the Fn binding sites, those who were homologus with it in other streptococcus played a major role in the process of infection so FBPS was assumed to be an important involving protein in the way of the adhesion of SS2to host.
Under this background, we used SS2high virulent strain SC19as the parental strain, take fbps as the study object, construct a fbps deletion mutant, and study its biological traits and virulence changes compare to the wild type strain. More detailed imformation about our research are listed below:
1. construction of temperature-sensitive recombination suicide plasmid
Use SS2high virulent strain SC19isolated from Sichuan province as the parental strain, PCR amplifies the780bp upstream sequnces and810bp downstream sequences. Then use fusion PCR to amplify the whole upstream and downstream fragment of fbps and link to the pMDx-18T vector to construct clone vector pMDx-18T-f. Use restriction enzeymes EcoRI and BamHI to digest both pMDx-18T-f and pSET4s then link them to construct the suicide plasmid pSET4s-fbps.
2. construction of the SS2fbps gene deletion mutant The suicide recombinat plasmid pSET4s-fbps was transformed by electroporation into the SS2wildtype strain SC19. Through a double seletion of temperature and antibiotics, select the fbps gene deletion mutant. Then use PCR to futher verify.
3. research of the SS2fbps gene deletion mutant's biological traits Study the genetic stability, in vitro growth and proliferation situation of the SS2fbps deletion mutant. The results showed that afer cultured more10generation the mutant was still stable. The growth curves found that the fbps deletion don't effect the growth of SS2in vivo.
4. the study of the effects of how fbps works when SS2adhesion, invation and do cytotoxicity to host cells
Use Hep-2cells to be the cell model, to compare the ability of adhesion and invasion of SC19and SS2fbps deletion mutant, evaluate the LDH concentration to compare the two strains'ability of cytotoxicity. The results showed tha the ability of SS2fbps deletion mutant adhesion to the Hep-2cells is apparently descent about78%, and the ability of invasion also decreased obviously about23%, test the cytotoxicity of mutant to cells compared with the wildtype SC19strain showed it decreased a lot about32%, too.
5. study the impact of deletion of fbps on the pathopoiesis
Use wildtype SC19and fbps deletion mutant intraperitoneal inject KM mice as animal models with the dosage of10cfu/ml,108cfu/ml and109cfu/ml, after injection observe the clinical symptoms each12h. The results showed that the death rate was much lower in the mutant injection group to the wildtype injection group. Dissect mice at different to screen the bacteria showed deletion of fbps leads to the decline of field planting. Histopathology observation showed that SS2fbps deletion mutant and wildtype SC19have the similar pathopoiesis areas, but the former has much lighter pathological change levels. The virulence of mutant is decreased.
SS2对宿主的粘附过程可能是其致病过程中一个关键的步骤。SS2和宿主细胞的粘附并非一个简单的过程,在细菌和细胞表面都有多种相关因子参与了此过程,因此研究在此过程中发挥作用的关键因子可能会对SS2致病机理阐释发挥重要作用。
猪链球菌2型基因组中的fbps基因编码了一种具有纤连蛋白结合位点的表面膜蛋白(fibronectin binding proteins, FBPS),和它具有同源性的同样具有Fn结合活性的蛋白在其他链球菌的感染中发挥着关键的作用,因此推断,FBPS可能是猪链球菌感染的吸附过程中的一个重要的参与蛋白。
在此背景之下,我们以SS2高致病性菌株SC19作为亲本菌株,以纤连蛋白结合蛋白编码基因作为研究对象,构建了fbps基因的缺失突变株,并在此基础上分析fbps基因的缺失对SS2基本生物学性状以及对致病力的影响,主要研究内容包括：
1温敏型自杀性重组质粒的构建
以2005年四川分离的SS2高致病性菌株SC19为亲本菌株,分别扩增fbps基因的780bp上游序列和81bp的下游序列,并利用融合PCR的方法获得上下游同源臂全长片段并连接到pMDx-18T载体,构建克隆载体pMDx-18T-f,分别用EcoR1和BamH1同时酶切pMDx-18T-f和pSET4s载体后连接,构建自杀性重组转移质粒pSET4s-fbps。
2纤连蛋白结合蛋白fbps基因突变株的构建
将重组质粒pSET4s-fbps电转化亲本菌株SC19感受态细胞,以壮观霉素抗性标记和温度敏感双重筛选,得到fbps基因缺失突变菌株。并进一步通过PCR等方式对筛选出的突变菌株进行验证。
3纤连蛋白结合蛋白fbps基因突变株生物学性状的研究
在构建的fbps基因突变株的基础上,研究缺失菌株的遗传稳定性,体外生长和增殖的能力等生物学性状。结果表明,连续传代十代突变株均能稳定遗传。野生株和突变株的生长没有显著的差异,fbps基因的缺失不会影响SS2的生长和繁殖的速度。
4fbps对宿主的吸附侵入以及毒性作用
以Hep-2细胞为模型,对比研究fbps缺失菌株和SC19野生型菌株粘附能力和侵入能力,以培养液中LDH的渗出为参照,测定培养液中LDH含量,比较突变株和野生菌株对细胞毒性作用。结果表明,fbps基因缺失突变株对Hep-2细胞的粘附能力和亲本株SC19相比下降了78%；对细胞侵入能力下降23%；测定LDH的浓度发现fbps基因突变株比野生型的毒性降低了32%。
5突变株致病性研究
将野生型菌株SC19和fbps基因缺失突变株分别以107cfu/ml,108cfu/ml和109cfu/ml的剂量腹腔注射昆明鼠(1m1)。结果显示,注射突变株的死亡率远远低于注射野生型SC19菌株的试验组。不同时间点剖杀小鼠不同器官的细菌动态分布表明,缺失了fbps基因导致细菌的组织定植能力显著下降。病理组织学观察发现SS2fbps基因缺失突变株和野生型在小鼠中病变范围类似,主要引起肺部,脑部和心脏的病变,但是突变株引起的病变程度较轻微,说明fbps基因缺失突变株致病性显著降低。
Streptococcus suis is an important pathogen of swine, and33serotypes have been identified based on their capsular antigen, of which the Streptococcus suis serotype2is the most important and frequently isolated from the clinical samples. SS2can cause a lot of serious diseases, such as meningitis, septicemia, arthritis, endocarditis, pnuemonia and even acute death in swine. SS2can also infect humans who were associated with pigs or swine industry directly; the serious cases would have STSS which leads to multiple organ failure or even death. It is an important zoonosis pathogen and is paid worldwide attention. Recent years several outbreaks of SS2have seriously harmed the swine industry and human health, then it was paid great attention by researchers of China or other countries.
The adhesion of SS2to the host maybe a key step in the pathopoiesis processes, so many works about adhesins have been carried out. But the adhesion of SS2to the host cells is not a simple way, many different components of SS2or the host cells were involved in it, so to find out which one play the major role in this process is usefully and necessarily to furter explain how SS2do harm to the hosts.
In the SS2genome there is a gene fbps encoding a surface membrane protein FBPS, which has the Fn binding sites, those who were homologus with it in other streptococcus played a major role in the process of infection so FBPS was assumed to be an important involving protein in the way of the adhesion of SS2to host.
Under this background, we used SS2high virulent strain SC19as the parental strain, take fbps as the study object, construct a fbps deletion mutant, and study its biological traits and virulence changes compare to the wild type strain. More detailed imformation about our research are listed below:
1. construction of temperature-sensitive recombination suicide plasmid
Use SS2high virulent strain SC19isolated from Sichuan province as the parental strain, PCR amplifies the780bp upstream sequnces and810bp downstream sequences. Then use fusion PCR to amplify the whole upstream and downstream fragment of fbps and link to the pMDx-18T vector to construct clone vector pMDx-18T-f. Use restriction enzeymes EcoRI and BamHI to digest both pMDx-18T-f and pSET4s then link them to construct the suicide plasmid pSET4s-fbps.
2. construction of the SS2fbps gene deletion mutant The suicide recombinat plasmid pSET4s-fbps was transformed by electroporation into the SS2wildtype strain SC19. Through a double seletion of temperature and antibiotics, select the fbps gene deletion mutant. Then use PCR to futher verify.
3. research of the SS2fbps gene deletion mutant's biological traits Study the genetic stability, in vitro growth and proliferation situation of the SS2fbps deletion mutant. The results showed that afer cultured more10generation the mutant was still stable. The growth curves found that the fbps deletion don't effect the growth of SS2in vivo.
4. the study of the effects of how fbps works when SS2adhesion, invation and do cytotoxicity to host cells
Use Hep-2cells to be the cell model, to compare the ability of adhesion and invasion of SC19and SS2fbps deletion mutant, evaluate the LDH concentration to compare the two strains'ability of cytotoxicity. The results showed tha the ability of SS2fbps deletion mutant adhesion to the Hep-2cells is apparently descent about78%, and the ability of invasion also decreased obviously about23%, test the cytotoxicity of mutant to cells compared with the wildtype SC19strain showed it decreased a lot about32%, too.
5. study the impact of deletion of fbps on the pathopoiesis
Use wildtype SC19and fbps deletion mutant intraperitoneal inject KM mice as animal models with the dosage of10cfu/ml,108cfu/ml and109cfu/ml, after injection observe the clinical symptoms each12h. The results showed that the death rate was much lower in the mutant injection group to the wildtype injection group. Dissect mice at different to screen the bacteria showed deletion of fbps leads to the decline of field planting. Histopathology observation showed that SS2fbps deletion mutant and wildtype SC19have the similar pathopoiesis areas, but the former has much lighter pathological change levels. The virulence of mutant is decreased.
引文
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