【摘要】：豬鏈球菌(Streptococcusssuis, SS)是一種革蘭氏陽性兼性厭氧球菌,根據(jù)其表面抗原莢膜多糖(CPS)的不同可分為33個(gè)血清型(1 -31,33及1/2)。豬鏈球菌2型(SS2)是世界范圍內(nèi)流行最廣、致病性最強(qiáng)的血清型,不僅能引起豬的敗血癥、關(guān)節(jié)炎、腦膜炎、心內(nèi)膜炎等多種疾病,還可以感染人,導(dǎo)致人的腦膜炎、敗血癥甚至死亡。然而,SS2的不同菌株毒力差異很大,而造成這種差異的物質(zhì)基礎(chǔ)—毒力因子,雖然對其開展的研究已經(jīng)進(jìn)行多年,但清晰的致病機(jī)理,以及不同毒力因子之間的相互關(guān)系尚不清楚。本論文針對SS2天然強(qiáng)弱毒株、人工致強(qiáng)致弱突變株以及體內(nèi)分離和體外培養(yǎng)的菌株三種不同來源的樣本,分別進(jìn)行了比較蛋白組學(xué)及蛋白相互作用網(wǎng)絡(luò)分析,研究差異表達(dá)蛋白對SS2毒力的影響。另外,利用修飾組學(xué)技術(shù),首次對SS2蛋白質(zhì)的翻譯后修飾,琥珀�；揎椉耙阴；揎椩赟S2生命活動中的作用進(jìn)行了初步的探索。1天然SS2強(qiáng)弱毒株的比較基因組學(xué)和比較蛋白組學(xué)分析SS2是一種重要的人畜共患病病原,但不同菌株之間存在毒力的差異。我們利用全基因組測序和同位素相對標(biāo)記與絕對定量技術(shù)(isobaric tag for relative and absolute quantitation, iTRAQ),對四川大暴發(fā)的代表菌株,天然強(qiáng)毒株ZY05719和天然弱毒株T15進(jìn)行了比較基因組學(xué)和比較蛋白組學(xué)分析。發(fā)現(xiàn)強(qiáng)毒株中特有的148個(gè)基因中有33個(gè)可以表達(dá)。結(jié)合操縱子預(yù)測、胞壁錨定基序(I/L)(P/A)XTG、酶活基序TL(L/V)TC等分析,發(fā)現(xiàn)這33個(gè)蛋白中存在分選酶(Sortase)系統(tǒng)。進(jìn)一步分析發(fā)現(xiàn)ZY05719基因組中共含有5種Sortase C ( C類分選酶,催化菌毛合成)和一種Sortase A ( A類分選酶,修飾表面蛋白)。其中三種編碼C類分選酶的基因僅存在于強(qiáng)毒基因組中,且屬于同一個(gè)操縱子,并分別共用堿基,這三個(gè)C類分選酶的編碼基因分別被命名為srtC1,srtC2和srtC3。在這三個(gè)C類分選酶的下游,緊鄰的操縱子中,通過iTRAQ表達(dá)量的差異及C類分選酶催化的底物特征找到這三個(gè)C類分選酶可能作用的底物,命名為C123。另外兩種C類分選酶,將其對應(yīng)基因命名為srtC4和srtC5。iTRAQ結(jié)果證明兩者在強(qiáng)毒株中的表達(dá)量要顯著高于弱毒株。同時(shí)發(fā)現(xiàn)Sortase C4的疑似底物,位于同一操縱子的C4,其在強(qiáng)毒株中的表達(dá)量顯著高于弱毒株。本章首次系統(tǒng)地鑒定了 SS2中僅存在的A類及C類分選酶系統(tǒng),并分析了酶與底物的對應(yīng)關(guān)系。后續(xù)分析發(fā)現(xiàn),srtC1,srtC2,srtC3僅廣泛存在于強(qiáng)毒株中,且敲除C類分選酶及其作用底物相關(guān)基因后,菌株毒力變?nèi)?證明其參與SS2毒力的形成。2人工構(gòu)建的強(qiáng)弱毒株的比較蛋白組學(xué)分析豬鏈球菌天然強(qiáng)弱毒株在基因組上存在較大的差異(如不同的SS2菌株中,基因組大小從2.0到2.24 Mb不等),基因組的差異映射到蛋白組上會造成更多的差異,給后續(xù)分析帶來困難。為了規(guī)避這個(gè)問題,我們以ZY05719為親本株構(gòu)建了一系列基因缺失株,經(jīng)動物實(shí)驗(yàn)分析發(fā)現(xiàn)了毒力減弱的△pepT突變株和毒力增強(qiáng)的△rfeA突變株。這兩株突變株的基因組背景高度一致,其蛋白表達(dá)水平的差異極有可能是導(dǎo)致毒力差異的主要原因之一。結(jié)合焚熒光差異雙向電泳2D-DIGE (two dimension difference gel electrophoresis)及蛋白質(zhì)非標(biāo)記定量技術(shù)(label-free)等適合比較背景較一致的樣本的蛋白質(zhì)組學(xué)技術(shù),以親本株ZY05719為對照,分析了 ApepT和△rfeA分泌蛋白的變化,共鑒定出38個(gè)差異蛋白,其中32個(gè)在△pepT中的表達(dá)量出現(xiàn)差異,17個(gè)在△rfeA中的表達(dá)量出現(xiàn)差異,11個(gè)在兩個(gè)菌株中都出現(xiàn)差異。差異蛋白共包括5個(gè)已知的毒力因子和33個(gè)新發(fā)現(xiàn)的毒力相關(guān)蛋白。為了研究這些新鑒定到的差異蛋白是否確實(shí)與毒力相關(guān),基于String數(shù)據(jù)庫和Cytoscape軟件,我們首次構(gòu)建出SS的毒力互作網(wǎng)絡(luò)。發(fā)現(xiàn)新型的差異蛋白在由已知的毒力因子形成的網(wǎng)絡(luò)中起到重要的橋梁作用。如新發(fā)現(xiàn)的EF-Tu等在毒力網(wǎng)絡(luò)中作為重要的節(jié)點(diǎn),很好地連接了新發(fā)現(xiàn)的差異蛋白和已知的毒力因子,更好地補(bǔ)充和完善了現(xiàn)有的毒力體系。其中一個(gè)新發(fā)現(xiàn)的差異蛋白SBP2,位于菌體表面,在毒力增強(qiáng)株中表達(dá)上調(diào),并牽涉在毒力網(wǎng)絡(luò)中。進(jìn)一步分析發(fā)現(xiàn)sbp2僅廣泛存在于SS2強(qiáng)毒株中。重組SBP2蛋白具有HEp-2細(xì)胞黏附性。Far-WB實(shí)驗(yàn)推測這種黏附作用部分依靠SBP2與宿主細(xì)胞外基質(zhì)的重要成分纖連蛋白(fibronectin, FN)和層黏連蛋白(laminin, LN)的結(jié)合完成。3體內(nèi)分離和體外培養(yǎng)的菌株的比較蛋白組學(xué)分析細(xì)菌在體外和體內(nèi)條件下的生長狀況是不同的,單純對體外培養(yǎng)條件下的細(xì)菌進(jìn)行研究并不能反映真實(shí)的細(xì)菌的生命狀態(tài)。利用豬鏈球菌強(qiáng)毒株ZY05719對易感豬進(jìn)行人工感染,并從感染豬的血液中分離體內(nèi)感染增殖的細(xì)菌,將它與體外培養(yǎng)基培養(yǎng)的細(xì)菌進(jìn)行比較蛋白組學(xué)分析,有利于認(rèn)識細(xì)菌進(jìn)入體內(nèi)后為存活及引起感染蛋白表達(dá)量發(fā)生的變化。由于體內(nèi)菌株含量較少,采用對蛋白量要求較低的iTRAQ進(jìn)行比較分析。共鑒定出188個(gè)差異蛋白,其中89個(gè)蛋白在體內(nèi)表達(dá)上調(diào),99個(gè)下調(diào)。后續(xù)重點(diǎn)對這些差異蛋白進(jìn)行操縱子、GO、KEGG通路和蛋白結(jié)構(gòu)域等分類、聚類多方面生物信息學(xué)分析。發(fā)現(xiàn)體內(nèi)上調(diào)表達(dá)的蛋白主要與氨基酸合成、糖代謝及結(jié)合活性等相關(guān),下調(diào)的蛋白則與脂肪酸合成代謝相關(guān)。通過構(gòu)建新發(fā)現(xiàn)的差異蛋白與已知的體內(nèi)感染相關(guān)的毒力因子的互作網(wǎng)絡(luò),發(fā)現(xiàn)大量差異蛋白與已知的體內(nèi)感染相關(guān)因子存在相互作用。其中最重要的上調(diào)蛋白,乙醇/乙醛脫氫酶,adhE(acetaldehyde-CoA/alcohol dehydrogenase)處于網(wǎng)絡(luò)中重要的節(jié)點(diǎn),不僅與細(xì)菌的厭氧呼吸有關(guān),也通過與宿主細(xì)胞(Caco-2)的Hsp60蛋白的互作介導(dǎo)細(xì)菌對宿主的黏附。敲除相關(guān)基因后,菌株對Caco-2細(xì)胞的黏附顯著下降。而通過使用抗體封閉下調(diào)通路中與脂肪酸合成代謝有關(guān)的蛋白fabG,可提高細(xì)菌在血液中的存活率。4 SS2全菌蛋白的乙�；揎椉扮牾；揎椯嚢彼岬孽；饔檬且环N重要的翻譯后修飾作用(PTM),包括乙酰化和琥珀�；�,與多種細(xì)胞進(jìn)程有關(guān)。不僅蛋白表達(dá)量的差異與細(xì)菌的毒力相關(guān),蛋白的翻譯后修飾也可能導(dǎo)致毒力的差異。有報(bào)道稱琥珀�；c布魯菌的毒力有關(guān),乙�；c沙門菌的毒力相關(guān)。但在豬鏈球菌中,賴氨酸�；饔玫墓δ苓�未被報(bào)道,本論文對SS中的賴氨酸琥珀�；鸵阴；@兩種�；饔眠M(jìn)行了第一次全面分析。結(jié)合親和富集和高分辨率LC-MS/MS分析的方法,共鑒定出SS2強(qiáng)毒株的51個(gè)蛋白的76個(gè)琥珀�；稽c(diǎn)(占總蛋白的2.6%)及888個(gè)蛋白的2901處賴氨酸乙�；揎椢稽c(diǎn)(占總蛋白的46.1%)。進(jìn)一步的蛋白注釋、功能分類、功能富集、基序分析、聚類分析等生物信息學(xué)分析發(fā)現(xiàn)這些發(fā)生修飾的蛋白參與各種細(xì)胞功能,尤其是在代謝、細(xì)胞進(jìn)程,催化、結(jié)合活性,核糖體相關(guān)的進(jìn)程中。兩種修飾之間存在廣泛的重疊作用。且發(fā)現(xiàn)多個(gè)已知的毒力因子同時(shí)發(fā)生兩種修飾作用,如MRP, GAPDH,enolase,5'-nuclcotidase和八ABC transporter等。但兩種修飾識別的基序不相同,且乙�；揎棸l(fā)揮作用的范圍更廣泛,也與轉(zhuǎn)運(yùn)、黏附、調(diào)控、信號轉(zhuǎn)導(dǎo)等相關(guān)。這一系統(tǒng)的分析為進(jìn)一步研究琥珀�；鸵阴；赟S生理及致病機(jī)制中所起的作用,奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.61

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本文編號：2140796