猪链球菌2型蛋白的差异表达及翻译后修饰对其毒力的影响
发布时间：2018-07-24 08:25
【摘要】：猪链球菌(Streptococcusssuis, SS)是一种革兰氏阳性兼性厌氧球菌,根据其表面抗原荚膜多糖(CPS)的不同可分为33个血清型(1 -31,33及1/2)。猪链球菌2型(SS2)是世界范围内流行最广、致病性最强的血清型,不仅能引起猪的败血症、关节炎、脑膜炎、心内膜炎等多种疾病,还可以感染人,导致人的脑膜炎、败血症甚至死亡。然而,SS2的不同菌株毒力差异很大,而造成这种差异的物质基础—毒力因子,虽然对其开展的研究已经进行多年,但清晰的致病机理,以及不同毒力因子之间的相互关系尚不清楚。本论文针对SS2天然强弱毒株、人工致强致弱突变株以及体内分离和体外培养的菌株三种不同来源的样本,分别进行了比较蛋白组学及蛋白相互作用网络分析,研究差异表达蛋白对SS2毒力的影响。另外,利用修饰组学技术,首次对SS2蛋白质的翻译后修饰,琥珀酰化修饰及乙酰化修饰在SS2生命活动中的作用进行了初步的探索。1天然SS2强弱毒株的比较基因组学和比较蛋白组学分析SS2是一种重要的人畜共患病病原,但不同菌株之间存在毒力的差异。我们利用全基因组测序和同位素相对标记与绝对定量技术(isobaric tag for relative and absolute quantitation, iTRAQ),对四川大暴发的代表菌株,天然强毒株ZY05719和天然弱毒株T15进行了比较基因组学和比较蛋白组学分析。发现强毒株中特有的148个基因中有33个可以表达。结合操纵子预测、胞壁锚定基序(I/L)(P/A)XTG、酶活基序TL(L/V)TC等分析,发现这33个蛋白中存在分选酶(Sortase)系统。进一步分析发现ZY05719基因组中共含有5种Sortase C ( C类分选酶,催化菌毛合成)和一种Sortase A ( A类分选酶,修饰表面蛋白)。其中三种编码C类分选酶的基因仅存在于强毒基因组中,且属于同一个操纵子,并分别共用碱基,这三个C类分选酶的编码基因分别被命名为srtC1,srtC2和srtC3。在这三个C类分选酶的下游,紧邻的操纵子中,通过iTRAQ表达量的差异及C类分选酶催化的底物特征找到这三个C类分选酶可能作用的底物,命名为C123。另外两种C类分选酶,将其对应基因命名为srtC4和srtC5。iTRAQ结果证明两者在强毒株中的表达量要显著高于弱毒株。同时发现Sortase C4的疑似底物,位于同一操纵子的C4,其在强毒株中的表达量显著高于弱毒株。本章首次系统地鉴定了 SS2中仅存在的A类及C类分选酶系统,并分析了酶与底物的对应关系。后续分析发现,srtC1,srtC2,srtC3仅广泛存在于强毒株中,且敲除C类分选酶及其作用底物相关基因后,菌株毒力变弱,证明其参与SS2毒力的形成。2人工构建的强弱毒株的比较蛋白组学分析猪链球菌天然强弱毒株在基因组上存在较大的差异(如不同的SS2菌株中,基因组大小从2.0到2.24 Mb不等),基因组的差异映射到蛋白组上会造成更多的差异,给后续分析带来困难。为了规避这个问题,我们以ZY05719为亲本株构建了一系列基因缺失株,经动物实验分析发现了毒力减弱的△pepT突变株和毒力增强的△rfeA突变株。这两株突变株的基因组背景高度一致,其蛋白表达水平的差异极有可能是导致毒力差异的主要原因之一。结合焚荧光差异双向电泳2D-DIGE (two dimension difference gel electrophoresis)及蛋白质非标记定量技术(label-free)等适合比较背景较一致的样本的蛋白质组学技术,以亲本株ZY05719为对照,分析了 ApepT和△rfeA分泌蛋白的变化,共鉴定出38个差异蛋白,其中32个在△pepT中的表达量出现差异,17个在△rfeA中的表达量出现差异,11个在两个菌株中都出现差异。差异蛋白共包括5个已知的毒力因子和33个新发现的毒力相关蛋白。为了研究这些新鉴定到的差异蛋白是否确实与毒力相关,基于String数据库和Cytoscape软件,我们首次构建出SS的毒力互作网络。发现新型的差异蛋白在由已知的毒力因子形成的网络中起到重要的桥梁作用。如新发现的EF-Tu等在毒力网络中作为重要的节点,很好地连接了新发现的差异蛋白和已知的毒力因子,更好地补充和完善了现有的毒力体系。其中一个新发现的差异蛋白SBP2,位于菌体表面,在毒力增强株中表达上调,并牵涉在毒力网络中。进一步分析发现sbp2仅广泛存在于SS2强毒株中。重组SBP2蛋白具有HEp-2细胞黏附性。Far-WB实验推测这种黏附作用部分依靠SBP2与宿主细胞外基质的重要成分纤连蛋白(fibronectin, FN)和层黏连蛋白(laminin, LN)的结合完成。3体内分离和体外培养的菌株的比较蛋白组学分析细菌在体外和体内条件下的生长状况是不同的,单纯对体外培养条件下的细菌进行研究并不能反映真实的细菌的生命状态。利用猪链球菌强毒株ZY05719对易感猪进行人工感染,并从感染猪的血液中分离体内感染增殖的细菌,将它与体外培养基培养的细菌进行比较蛋白组学分析,有利于认识细菌进入体内后为存活及引起感染蛋白表达量发生的变化。由于体内菌株含量较少,采用对蛋白量要求较低的iTRAQ进行比较分析。共鉴定出188个差异蛋白,其中89个蛋白在体内表达上调,99个下调。后续重点对这些差异蛋白进行操纵子、GO、KEGG通路和蛋白结构域等分类、聚类多方面生物信息学分析。发现体内上调表达的蛋白主要与氨基酸合成、糖代谢及结合活性等相关,下调的蛋白则与脂肪酸合成代谢相关。通过构建新发现的差异蛋白与已知的体内感染相关的毒力因子的互作网络,发现大量差异蛋白与已知的体内感染相关因子存在相互作用。其中最重要的上调蛋白,乙醇/乙醛脱氢酶,adhE(acetaldehyde-CoA/alcohol dehydrogenase)处于网络中重要的节点,不仅与细菌的厌氧呼吸有关,也通过与宿主细胞(Caco-2)的Hsp60蛋白的互作介导细菌对宿主的黏附。敲除相关基因后,菌株对Caco-2细胞的黏附显著下降。而通过使用抗体封闭下调通路中与脂肪酸合成代谢有关的蛋白fabG,可提高细菌在血液中的存活率。4 SS2全菌蛋白的乙酰化修饰及琥珀酰化修饰赖氨酸的酰化作用是一种重要的翻译后修饰作用(PTM),包括乙酰化和琥珀酰化等,与多种细胞进程有关。不仅蛋白表达量的差异与细菌的毒力相关,蛋白的翻译后修饰也可能导致毒力的差异。有报道称琥珀酰化与布鲁菌的毒力有关,乙酰化与沙门菌的毒力相关。但在猪链球菌中,赖氨酸酰化作用的功能还未被报道,本论文对SS中的赖氨酸琥珀酰化和乙酰化这两种酰基化作用进行了第一次全面分析。结合亲和富集和高分辨率LC-MS/MS分析的方法,共鉴定出SS2强毒株的51个蛋白的76个琥珀酰化位点(占总蛋白的2.6%)及888个蛋白的2901处赖氨酸乙酰化修饰位点(占总蛋白的46.1%)。进一步的蛋白注释、功能分类、功能富集、基序分析、聚类分析等生物信息学分析发现这些发生修饰的蛋白参与各种细胞功能,尤其是在代谢、细胞进程,催化、结合活性,核糖体相关的进程中。两种修饰之间存在广泛的重叠作用。且发现多个已知的毒力因子同时发生两种修饰作用,如MRP, GAPDH,enolase,5'-nuclcotidase和八ABC transporter等。但两种修饰识别的基序不相同,且乙酰化修饰发挥作用的范围更广泛,也与转运、黏附、调控、信号转导等相关。这一系统的分析为进一步研究琥珀酰化和乙酰化在SS生理及致病机制中所起的作用,奠定了基础。
[Abstract]:Streptococcus suis (Streptococcusssuis, SS) is a gram positive facultative anaerobes, which can be divided into 33 serotypes (1 -31,33 and 1/2) according to the difference of its surface antigen capsular polysaccharide (CPS). Streptococcus suis type 2 (SS2) is the most prevalent and most pathogenic serotype in the world. It can not only cause septicemia, arthritis, and meningitis, but also the most prevalent serotypes in the world. A variety of diseases such as endocarditis can also infect people, cause meningitis, septicemia and even death. However, the virulence of different strains of SS2 is very different, and the material base of this difference is toxic factor, although the research on it has been carried out for many years, but clear pathogenic mechanism, and the interaction of different virulence factors. The relationship is not yet clear. In this paper, three samples of SS2 natural strong and weak strains, artificially induced weak mutants and isolated and cultured strains in vivo were used to compare proteomics and protein interaction network analysis to study the effect of differentially expressed proteins on the virulence of SS2. For the first time the post-translational modification of SS2 protein, succinylation and acetylation modification in the SS2 life activities were preliminarily explored in the comparative genomics and comparative proteomic analysis of.1 SS2 strong and weak strains, SS2 is an important zoonosis pathogen, but the differences of virulence between different strains are used. Complete genome sequencing and isotopic relative labeling and absolute quantification (isobaric tag for relative and absolute quantitation, iTRAQ), comparative genomics and comparative proteomics analysis of the representative strains of the large outbreak in Sichuan, the natural strong strain ZY05719 and the natural weak strain T15, were analyzed. The 148 specific genes in the strong virulent strains were found. 33 can be expressed. Combined with the analysis of operon prediction, I/L (P/A) XTG, TL (L/V) TC, and so on, the separation enzyme (Sortase) system is found in the 33 proteins. Further analysis found that the ZY05719 genome contains 5 Sortase C (C class separation enzyme, catalytic pili synthesis) and a kind of class a sorting enzyme, modification Three kinds of genes encoding C class sorting enzymes exist only in the virulent genomes, and they belong to the same operon and share their bases respectively. The encoding genes of the three C sorting enzymes are named srtC1, srtC2 and srtC3. are downstream of the three C sorting enzymes, and the difference in the iTRAQ expression in the adjacent operon is different and The substrate characteristics of the C class separation enzyme catalyze the substrate of the three C sorting enzymes, named as C123. and two other C sorting enzymes. Their corresponding genes are named srtC4 and srtC5.iTRAQ. The results show that the expression of both of them in the virulent strains is significantly higher than that of the weak strains. Meanwhile, the suspected substrate of Sortase C4 is located in the same operon. The expression of C4 in the strong virulent strain was significantly higher than that of the weak strain. This chapter systematically identified the only class A and the C sorting enzyme system in SS2 and analyzed the corresponding relationship between the enzyme and the substrate. The subsequent analysis found that srtC1, srtC2, srtC3 were only widely existed in the virulent strains and knocked out the C sorting enzyme and its action on the substrate related genes. The virulence of the strain is weak, which proves that it participates in the formation of the SS2 virulence of the strong and weak strains constructed by.2. Analysis of the genome of Streptococcus suis natural strong and weak strains (for example, different SS2 strains, the genome size varies from 2 to 2.24 Mb), the difference in the gene group mapping to the protein group will cause more difference. In order to avoid this problem, in order to avoid this problem, we constructed a series of gene deletion strains with ZY05719 as the parent strain, and found the delta pepT mutant with weakened virulence and the virulence enhanced Delta rfeA mutant by animal experiment. The two mutant strains have the same genome backview, and the difference of the protein expression level is extremely different. It may be one of the main causes of the difference in virulence. The proteomics technology, which is suitable for comparison of samples with relatively consistent background, is used in combination with 2D-DIGE (two dimension difference gel electrophoresis) and protein unmarked quantitative technique (label-free), which is a comparison of the parent strain ZY05719 as the control. ApepT and delta R are analyzed. The changes in feA secretory protein showed that 38 differential proteins were identified, 32 of which were expressed in Delta pepT, 17 in Delta rfeA and 11 in two strains. The difference proteins included 5 known virulence factors and 33 newly emerged virulence related proteins. Whether the difference proteins are really related to virulence, based on the String database and Cytoscape software, we first constructed the SS's virulence interaction network. It is found that the new differential protein plays an important bridge role in the network of known virulence factors. The newly discovered EF-Tu is an important node in the virulence network. The newly discovered differential proteins and known virulence factors have been well connected to complement and improve the existing virulence systems. One of the newly discovered differential proteins, SBP2, is located on the surface of the fungus, up-regulated in the virulence enhancement strain and involved in the virulence network. Further segregation analysis found that SBP2 is only widely existed in the SS2 strains. Group SBP2 protein has HEp-2 cell adhesion.Far-WB experiment to speculate that the adhesion function partly relies on the combination of SBP2 and the important component of the extracellular matrix of the host extracellular matrix (fibronectin, FN) and laminin (laminin, LN) to complete the comparative proteomics of the isolated and cultured strains of.3 in vitro and in vitro culture, the bacteria in vitro and in vivo The growth condition is different. The study of bacteria under culture in vitro can not reflect the life state of the true bacteria. Using Streptococcus suis strain ZY05719 to infect the susceptible pigs, and isolate the infected bacteria from the blood of the infected pigs, and enter the bacteria in the culture medium in vitro. Comparative proteomics analysis was helpful to understand the changes in the survival and infection protein expression in the body after the bacteria entered the body. Compared with the low protein content of iTRAQ, 188 different proteins were identified, 89 of them were up regulated in the body and 99 down regulated. These differentially expressed proteins are classified by operon, GO, KEGG pathway and protein domain. It is found that the up-regulated proteins in the body are mainly related to amino acid synthesis, sugar metabolism and binding activity, and the down regulated proteins are associated with fatty acids. A known interaction network of virulence factors associated with infection in the body has found that a large number of differential proteins interact with known internal infection related factors. The most important up - regulated protein, ethanol / aldehyde dehydrogenase, adhE (acetaldehyde-CoA/alcohol dehydrogenase) is an important node in the network, not only with the anaerobic respiration of bacteria. The adhesion of bacteria to the host is mediated by interaction with the Hsp60 protein of the host cell (Caco-2). After knocking out the related genes, the adhesion of the strain to Caco-2 cells decreases significantly. By using the antibody to close down the protein fabG associated with fatty acid synthesis in the pathway, the survival rate of bacteria in the blood can be increased by.4 SS2 whole bacteria protein. Acetylation and succinylation modification of lysine is an important post-translational modification (PTM), including acetylation and succinylation, which is related to a variety of cell processes. The difference in protein expression is related to the virulence of the bacteria, and the post-translational modification of the protein may also lead to the difference of virulence. Acylation is related to the virulence of Brucella, and acetylation is related to the virulence of Salmonella. But in Streptococcus suis, the function of lysine acylation has not been reported. The first comprehensive analysis of the two acylation effects of lysine succinylation and acetylation in SS was carried out in this paper. Methods a total of 76 succinylation sites (2.6% of total protein, 2.6% of the total protein) and 2901 lysine acetylation sites (46.1% of total protein) of the 51 proteins of the SS2 strain were identified. Further bioinformatics analysis, such as protein annotation, functional classification, functional enrichment, motif analysis, polyclass analysis, and other bioinformatics analysis found these modifications Protein participates in a variety of cell functions, especially in metabolism, cell process, catalysis, binding activity, and ribosome related processes. There are extensive overlaps between the two modifications. Several known virulence factors have two kinds of modification, such as MRP, GAPDH, enolase, 5'-nuclcotidase and eight ABC transporter. The basis for further study of the role of succinylation and acetylation in the SS physiology and pathogeny mechanisms of succinylation and acetylation is established.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.61
[Abstract]:Streptococcus suis (Streptococcusssuis, SS) is a gram positive facultative anaerobes, which can be divided into 33 serotypes (1 -31,33 and 1/2) according to the difference of its surface antigen capsular polysaccharide (CPS). Streptococcus suis type 2 (SS2) is the most prevalent and most pathogenic serotype in the world. It can not only cause septicemia, arthritis, and meningitis, but also the most prevalent serotypes in the world. A variety of diseases such as endocarditis can also infect people, cause meningitis, septicemia and even death. However, the virulence of different strains of SS2 is very different, and the material base of this difference is toxic factor, although the research on it has been carried out for many years, but clear pathogenic mechanism, and the interaction of different virulence factors. The relationship is not yet clear. In this paper, three samples of SS2 natural strong and weak strains, artificially induced weak mutants and isolated and cultured strains in vivo were used to compare proteomics and protein interaction network analysis to study the effect of differentially expressed proteins on the virulence of SS2. For the first time the post-translational modification of SS2 protein, succinylation and acetylation modification in the SS2 life activities were preliminarily explored in the comparative genomics and comparative proteomic analysis of.1 SS2 strong and weak strains, SS2 is an important zoonosis pathogen, but the differences of virulence between different strains are used. Complete genome sequencing and isotopic relative labeling and absolute quantification (isobaric tag for relative and absolute quantitation, iTRAQ), comparative genomics and comparative proteomics analysis of the representative strains of the large outbreak in Sichuan, the natural strong strain ZY05719 and the natural weak strain T15, were analyzed. The 148 specific genes in the strong virulent strains were found. 33 can be expressed. Combined with the analysis of operon prediction, I/L (P/A) XTG, TL (L/V) TC, and so on, the separation enzyme (Sortase) system is found in the 33 proteins. Further analysis found that the ZY05719 genome contains 5 Sortase C (C class separation enzyme, catalytic pili synthesis) and a kind of class a sorting enzyme, modification Three kinds of genes encoding C class sorting enzymes exist only in the virulent genomes, and they belong to the same operon and share their bases respectively. The encoding genes of the three C sorting enzymes are named srtC1, srtC2 and srtC3. are downstream of the three C sorting enzymes, and the difference in the iTRAQ expression in the adjacent operon is different and The substrate characteristics of the C class separation enzyme catalyze the substrate of the three C sorting enzymes, named as C123. and two other C sorting enzymes. Their corresponding genes are named srtC4 and srtC5.iTRAQ. The results show that the expression of both of them in the virulent strains is significantly higher than that of the weak strains. Meanwhile, the suspected substrate of Sortase C4 is located in the same operon. The expression of C4 in the strong virulent strain was significantly higher than that of the weak strain. This chapter systematically identified the only class A and the C sorting enzyme system in SS2 and analyzed the corresponding relationship between the enzyme and the substrate. The subsequent analysis found that srtC1, srtC2, srtC3 were only widely existed in the virulent strains and knocked out the C sorting enzyme and its action on the substrate related genes. The virulence of the strain is weak, which proves that it participates in the formation of the SS2 virulence of the strong and weak strains constructed by.2. Analysis of the genome of Streptococcus suis natural strong and weak strains (for example, different SS2 strains, the genome size varies from 2 to 2.24 Mb), the difference in the gene group mapping to the protein group will cause more difference. In order to avoid this problem, in order to avoid this problem, we constructed a series of gene deletion strains with ZY05719 as the parent strain, and found the delta pepT mutant with weakened virulence and the virulence enhanced Delta rfeA mutant by animal experiment. The two mutant strains have the same genome backview, and the difference of the protein expression level is extremely different. It may be one of the main causes of the difference in virulence. The proteomics technology, which is suitable for comparison of samples with relatively consistent background, is used in combination with 2D-DIGE (two dimension difference gel electrophoresis) and protein unmarked quantitative technique (label-free), which is a comparison of the parent strain ZY05719 as the control. ApepT and delta R are analyzed. The changes in feA secretory protein showed that 38 differential proteins were identified, 32 of which were expressed in Delta pepT, 17 in Delta rfeA and 11 in two strains. The difference proteins included 5 known virulence factors and 33 newly emerged virulence related proteins. Whether the difference proteins are really related to virulence, based on the String database and Cytoscape software, we first constructed the SS's virulence interaction network. It is found that the new differential protein plays an important bridge role in the network of known virulence factors. The newly discovered EF-Tu is an important node in the virulence network. The newly discovered differential proteins and known virulence factors have been well connected to complement and improve the existing virulence systems. One of the newly discovered differential proteins, SBP2, is located on the surface of the fungus, up-regulated in the virulence enhancement strain and involved in the virulence network. Further segregation analysis found that SBP2 is only widely existed in the SS2 strains. Group SBP2 protein has HEp-2 cell adhesion.Far-WB experiment to speculate that the adhesion function partly relies on the combination of SBP2 and the important component of the extracellular matrix of the host extracellular matrix (fibronectin, FN) and laminin (laminin, LN) to complete the comparative proteomics of the isolated and cultured strains of.3 in vitro and in vitro culture, the bacteria in vitro and in vivo The growth condition is different. The study of bacteria under culture in vitro can not reflect the life state of the true bacteria. Using Streptococcus suis strain ZY05719 to infect the susceptible pigs, and isolate the infected bacteria from the blood of the infected pigs, and enter the bacteria in the culture medium in vitro. Comparative proteomics analysis was helpful to understand the changes in the survival and infection protein expression in the body after the bacteria entered the body. Compared with the low protein content of iTRAQ, 188 different proteins were identified, 89 of them were up regulated in the body and 99 down regulated. These differentially expressed proteins are classified by operon, GO, KEGG pathway and protein domain. It is found that the up-regulated proteins in the body are mainly related to amino acid synthesis, sugar metabolism and binding activity, and the down regulated proteins are associated with fatty acids. A known interaction network of virulence factors associated with infection in the body has found that a large number of differential proteins interact with known internal infection related factors. The most important up - regulated protein, ethanol / aldehyde dehydrogenase, adhE (acetaldehyde-CoA/alcohol dehydrogenase) is an important node in the network, not only with the anaerobic respiration of bacteria. The adhesion of bacteria to the host is mediated by interaction with the Hsp60 protein of the host cell (Caco-2). After knocking out the related genes, the adhesion of the strain to Caco-2 cells decreases significantly. By using the antibody to close down the protein fabG associated with fatty acid synthesis in the pathway, the survival rate of bacteria in the blood can be increased by.4 SS2 whole bacteria protein. Acetylation and succinylation modification of lysine is an important post-translational modification (PTM), including acetylation and succinylation, which is related to a variety of cell processes. The difference in protein expression is related to the virulence of the bacteria, and the post-translational modification of the protein may also lead to the difference of virulence. Acylation is related to the virulence of Brucella, and acetylation is related to the virulence of Salmonella. But in Streptococcus suis, the function of lysine acylation has not been reported. The first comprehensive analysis of the two acylation effects of lysine succinylation and acetylation in SS was carried out in this paper. Methods a total of 76 succinylation sites (2.6% of total protein, 2.6% of the total protein) and 2901 lysine acetylation sites (46.1% of total protein) of the 51 proteins of the SS2 strain were identified. Further bioinformatics analysis, such as protein annotation, functional classification, functional enrichment, motif analysis, polyclass analysis, and other bioinformatics analysis found these modifications Protein participates in a variety of cell functions, especially in metabolism, cell process, catalysis, binding activity, and ribosome related processes. There are extensive overlaps between the two modifications. Several known virulence factors have two kinds of modification, such as MRP, GAPDH, enolase, 5'-nuclcotidase and eight ABC transporter. The basis for further study of the role of succinylation and acetylation in the SS physiology and pathogeny mechanisms of succinylation and acetylation is established.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.61
【参考文献】
相关期刊论文 前10条
1 杜德超;于岩飞;马彩凤;姚火春;陆承平;张炜;;猪链球菌2型不同mrp基因型与毒力的关系[J];微生物学通报;2016年01期
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