猪链球菌2型srtBCD菌毛簇基因功能初步研究
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摘要
猪链球菌(Streptococcus suis serotype , S. suis)是一种重要的人兽共患病病原菌。根据其表面荚膜多糖的抗原性,可以分为1-34和1/2共35个血清型,其中S. suis 2的致病性最强,临床检出率最高,给全世界的养猪业造成巨大的经济损失。尤其是近年来多次出现S.suis 2感染人事件,引起业界人士的高度关注。菌毛是革兰阳性病原菌与宿主细胞交互作用的重要功能结构,其中次要结构蛋白可能与病原菌的组织细胞亲嗜性相关,在细菌粘附、定植和侵袭过程发挥关键作用。和革兰阴性菌(G—)相比,革兰阳性(G+)菌菌毛编码基因与转肽酶sortase(srt)基因成簇存在,Srt催化菌毛单体共价交叉连接,最后锚定到细胞壁上。此外,值得关注的是,化脓链球菌(GAS)和无乳链球菌(GBS)的菌毛主要结构抗原还被证实是理想的疫苗侯选分子。通过对国内S. suis 2强毒株全基因组注释分析,我们发现两个在结构特征上与已知革兰阳性病原菌菌毛编码基因完全一致的基因簇,分别编码一组Sortase转肽酶和与已知菌毛结构蛋白同源的膜蛋白,命名为srtBCD和srtF菌毛簇,其中srtBCD簇仅存在于强毒株中。本研究以srtBCD菌毛簇为主要研究对象,同时展开对SrtF转肽酶的生物学功能研究,具体内容如下:
1 srtBCD菌毛簇SSU2099基因的克隆表达与免疫保护性
选取S.suis 2 srtBCD菌毛簇上菌毛辅助亚基编码基因SSU2099,PCR扩增目的片段,克隆入表达载体pET32a中原核表达,亲和层析法纯化重组蛋白。ELISA结果显示重组蛋白能够刺激小鼠产生高效价的免疫抗体,动物保护性实验表明该蛋白具有良好的免疫保护作用,提示菌毛亚基编码基因SSU2099是重要的疫苗候选分子。
2 srtBCD基因突变株的构建
利用同源重组原理构建中间为壮观霉素(SPC)、两侧为srtBCD基因上下游片段的重组质粒,将构建好的质粒电转化入S.suis 2感受态,筛选srtBCD缺失的突变株,并通过组合PCR和逆转录PCR对其进行验证。
3突变株?srtBCD的生物学功能研究
体外实验结果显示突变株ΔsrtBCD经反复传代培养后可以稳定生长、ΔsrtBCD突变株在对数期的生长速率明显减缓,且稳定期OD_(600)最大值小于野生株。粘附实验结果表明,ΔsrtBCD突变株对人类喉癌上皮细胞的粘附能力明显减弱,小鼠经05ZYH33野生株和突变株ΔsrtBCD攻毒后,致死率差异不具有统计学意义。推测srtBCD菌毛簇很可能编码S. suis 2表面的菌毛样结构,结果仍需通过免疫电镜及免疫印迹进一步验证。
4突变株?srtF的生物学功能研究
05ZYH33野生型和ΔsrtF突变株在菌落形态和溶血活性方面均无明显差异,但ΔsrtF突变株比野生型在对数初期的生长速率有增快的趋势。小鼠经05ZYH33野生型和ΔsrtF突变株攻毒后,致死率差异不具有统计学意义,结果和国外学者报导一致,证实S. suis 2中srtF菌毛簇编码的菌毛结构并非细菌致病作用必须的毒力因子。
Streptococcus suis 2 (S. suis 2) is an emerging zoonotic pathogen responsible for a wide range of life-threatening diseases in pigs and humans. To date, thirty-five serotypes (types 1/2, and 1-34) of S. suis have been described, of which S. suis 2 is the most frequently isolated and associated with disease. Various cell-surface multisubunit protein polymers, known as pili or fimbriae, have a pivotal role in the colonization of specific host tissues by many pathogenic bacteria. In contrast to Gram-negative bacteria, Gram-positive bacteria assemble pili by a distinct mechanism involving a transpeptidase called sortase. Sortase cross-links individual pilin monomers and ultimately joins the resulting covalent polymer to the cell-wall peptidoglycan. Most importantly, pilins confer a significant protection, which indicated that it can serve as a novel vaccine candidate. Based on bioinformatics analysis of the whole genome of S. suis 2 Chinese high virulent strain 05ZYH33, we found two gene clusters similar to pilus-associated gene clusters of some Gram-positive bacteria in genetic organization phenomena, designated srtBCD and srtF pilus clusters, which srtBCD cluster exists only in the virulent strain. In this study,? we characterized a functional member of srtBCD clusters and SrtF from a Chinese S. suis 2 isolate, 05ZYH33, And the following experiments are conducted:
1 Molecular cloning and immunological characterization of SSU2099 gene from srtBCD pilus island of Streptococcus suis 2
An open reading fragment, SSU2099, encoding pilus gene from srtBCD pilus island was identified based on bioinformatics analysis of the genome sequence of 05ZYH33 and alignment with relative protein family. The target DNA fragment was amplified using a pair of primers specific to SSU2099 gene. SSU2099 gene was cloned into prokaryotic expression plasmid pET32a subsequently, and the recombinant protein was expressed and purified. The mice anti-SSU2099 serum was obtained by immunizing mice with recombinant SSU2099 protein, and the titer of the serum was analyzed by ELISA. Animal test showed that vaccinating mice with recombinant SSU2099 confer a significant protection, which indicated that SSU2099 can serve as a novel vaccine candidate.
2 Construction the ?srtBCD mutant
To obtain the isogenic mutant srtBCD, the competent cells of 05ZYH33 were subjected to electrotrans formation with recombinant plasmid based on the principle of homologous recombination. The resulting mutant strains was further confirmed by a series of PCR and reverse transcription PCR.
3 Biological functions of ?srtBCD mutant
We demonstrated that mutant srtBCD reduced capacity to adhere to human laryngeal epithelial cell line compared to the wild-type strain. However, disruption of srtBCD had less effect on the virulence of S.suis in a mouse intraperitoneal model of infection. These results indicated that surface proteins anchored by SrtBCD are required for a normal level of bacterial binding. However, other factors may also be important for S.suis 2 virulence and interactions with host tissues.
4 Biological functions of ?srtF mutant
The resulting mutant strain exhibited growth kinetics equivalent to those of the WT parent strain upon cultivation in standard laboratory used in our in vitro assays. Abolishment of the expression of srtF did not result in impaired interactions of S.suis 2 with human laryngeal epithelial cell line. Furthermore, srtF mutant were as virulent as the wild type strain when evaluated in a murine model of S.suis . Taken together, these results suggest that srtF cluster in fact not critical for the full virulence of S. suis 2.
1 srtBCD菌毛簇SSU2099基因的克隆表达与免疫保护性
选取S.suis 2 srtBCD菌毛簇上菌毛辅助亚基编码基因SSU2099,PCR扩增目的片段,克隆入表达载体pET32a中原核表达,亲和层析法纯化重组蛋白。ELISA结果显示重组蛋白能够刺激小鼠产生高效价的免疫抗体,动物保护性实验表明该蛋白具有良好的免疫保护作用,提示菌毛亚基编码基因SSU2099是重要的疫苗候选分子。
2 srtBCD基因突变株的构建
利用同源重组原理构建中间为壮观霉素(SPC)、两侧为srtBCD基因上下游片段的重组质粒,将构建好的质粒电转化入S.suis 2感受态,筛选srtBCD缺失的突变株,并通过组合PCR和逆转录PCR对其进行验证。
3突变株?srtBCD的生物学功能研究
体外实验结果显示突变株ΔsrtBCD经反复传代培养后可以稳定生长、ΔsrtBCD突变株在对数期的生长速率明显减缓,且稳定期OD_(600)最大值小于野生株。粘附实验结果表明,ΔsrtBCD突变株对人类喉癌上皮细胞的粘附能力明显减弱,小鼠经05ZYH33野生株和突变株ΔsrtBCD攻毒后,致死率差异不具有统计学意义。推测srtBCD菌毛簇很可能编码S. suis 2表面的菌毛样结构,结果仍需通过免疫电镜及免疫印迹进一步验证。
4突变株?srtF的生物学功能研究
05ZYH33野生型和ΔsrtF突变株在菌落形态和溶血活性方面均无明显差异,但ΔsrtF突变株比野生型在对数初期的生长速率有增快的趋势。小鼠经05ZYH33野生型和ΔsrtF突变株攻毒后,致死率差异不具有统计学意义,结果和国外学者报导一致,证实S. suis 2中srtF菌毛簇编码的菌毛结构并非细菌致病作用必须的毒力因子。
Streptococcus suis 2 (S. suis 2) is an emerging zoonotic pathogen responsible for a wide range of life-threatening diseases in pigs and humans. To date, thirty-five serotypes (types 1/2, and 1-34) of S. suis have been described, of which S. suis 2 is the most frequently isolated and associated with disease. Various cell-surface multisubunit protein polymers, known as pili or fimbriae, have a pivotal role in the colonization of specific host tissues by many pathogenic bacteria. In contrast to Gram-negative bacteria, Gram-positive bacteria assemble pili by a distinct mechanism involving a transpeptidase called sortase. Sortase cross-links individual pilin monomers and ultimately joins the resulting covalent polymer to the cell-wall peptidoglycan. Most importantly, pilins confer a significant protection, which indicated that it can serve as a novel vaccine candidate. Based on bioinformatics analysis of the whole genome of S. suis 2 Chinese high virulent strain 05ZYH33, we found two gene clusters similar to pilus-associated gene clusters of some Gram-positive bacteria in genetic organization phenomena, designated srtBCD and srtF pilus clusters, which srtBCD cluster exists only in the virulent strain. In this study,? we characterized a functional member of srtBCD clusters and SrtF from a Chinese S. suis 2 isolate, 05ZYH33, And the following experiments are conducted:
1 Molecular cloning and immunological characterization of SSU2099 gene from srtBCD pilus island of Streptococcus suis 2
An open reading fragment, SSU2099, encoding pilus gene from srtBCD pilus island was identified based on bioinformatics analysis of the genome sequence of 05ZYH33 and alignment with relative protein family. The target DNA fragment was amplified using a pair of primers specific to SSU2099 gene. SSU2099 gene was cloned into prokaryotic expression plasmid pET32a subsequently, and the recombinant protein was expressed and purified. The mice anti-SSU2099 serum was obtained by immunizing mice with recombinant SSU2099 protein, and the titer of the serum was analyzed by ELISA. Animal test showed that vaccinating mice with recombinant SSU2099 confer a significant protection, which indicated that SSU2099 can serve as a novel vaccine candidate.
2 Construction the ?srtBCD mutant
To obtain the isogenic mutant srtBCD, the competent cells of 05ZYH33 were subjected to electrotrans formation with recombinant plasmid based on the principle of homologous recombination. The resulting mutant strains was further confirmed by a series of PCR and reverse transcription PCR.
3 Biological functions of ?srtBCD mutant
We demonstrated that mutant srtBCD reduced capacity to adhere to human laryngeal epithelial cell line compared to the wild-type strain. However, disruption of srtBCD had less effect on the virulence of S.suis in a mouse intraperitoneal model of infection. These results indicated that surface proteins anchored by SrtBCD are required for a normal level of bacterial binding. However, other factors may also be important for S.suis 2 virulence and interactions with host tissues.
4 Biological functions of ?srtF mutant
The resulting mutant strain exhibited growth kinetics equivalent to those of the WT parent strain upon cultivation in standard laboratory used in our in vitro assays. Abolishment of the expression of srtF did not result in impaired interactions of S.suis 2 with human laryngeal epithelial cell line. Furthermore, srtF mutant were as virulent as the wild type strain when evaluated in a murine model of S.suis . Taken together, these results suggest that srtF cluster in fact not critical for the full virulence of S. suis 2.
引文
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