一、鼠疫菌毒力相關(guān)蛋白與人蛋白相互作用的初步研究 二、鼠疫菌Ⅲ型分泌系統(tǒng)內(nèi)蛋白相互作用及LcrG調(diào)控的研究
發(fā)布時間：2018-07-23 11:05
【摘要】： 鼠疫是由鼠疫耶爾森氏菌引發(fā)的的一種烈性傳染病,歷史上曾經(jīng)發(fā)生三次人間鼠疫的世界大流行。鼠疫菌的致病機制依賴于它和宿主之間的相互作用而實現(xiàn)。揭示鼠疫菌與人之間的蛋白相互作用是深入了解其致病機制的重要基礎(chǔ),同時為研究有效的預(yù)防和控制鼠疫的發(fā)生和傳播的手段提供線索。本研究包括三部分:首先,采用高通量的酵母雙雜交技術(shù)(Y2H),以推測的鼠疫菌毒力相關(guān)蛋白為誘餌篩選人脾臟cDNA文庫,旨在獲得鼠疫菌與人之間蛋白相互作用的初步網(wǎng)絡(luò),并且獲得一批重要的蛋白相互作用(PPI),為深入揭示鼠疫菌的致病機制提供研究靶標;其次,應(yīng)用Y2H陣列篩選技術(shù)篩選鼠疫菌III型分泌系統(tǒng)(T3SS)內(nèi)部蛋白質(zhì)間的相互作用,為進一步闡明T3SS的結(jié)構(gòu)和功能奠定基礎(chǔ);第三,為進一步了解負調(diào)控因子LcrG在鼠疫菌T3SS基因表達中的作用,應(yīng)用全基因組芯片進行了鼠疫菌野生株和lcrG突變株的比較轉(zhuǎn)錄譜學(xué)研究。 第一部分:鼠疫菌毒力相關(guān)蛋白與人蛋白質(zhì)相互作用的研究 我們根據(jù)鼠疫菌和假結(jié)核菌的比較基因組學(xué)研究結(jié)果和本實驗室開展的鼠疫菌在不同刺激條件下的表達譜、血清抗體譜、蛋白芯片篩選的試驗結(jié)果,參考文獻中鼠疫菌相關(guān)研究的最新進展最終確定可能與人蛋白相互作用的鼠疫菌蛋白152個(占鼠疫菌整個基因組的3.75%)。利用高通量Y2H技術(shù)篩選鼠疫菌與人之間的PPI。本研究采用基于Gateway重組技術(shù)的ProQuest? Two-Hybrid System,便于高通量平行操作。通過Gateway重組技術(shù),成功構(gòu)建了154個誘餌載體,對應(yīng)于152個鼠疫菌蛋白的ORFs。其中存在自激活的誘餌有3個,分別是:YPCD1.26c,YPCD1.31c和YPMT1.61c。 通過順序轉(zhuǎn)化法進行規(guī)�；痀2H篩選,共對151個無自激活活性的誘餌至少篩選一次人脾臟cDNA文庫。篩選平板上長出的候選克隆分別通過生長實驗鑒定HIS和ADE報告基因表型,X-Gal分析鑒定lacZ報告基因表型。對得到的陽性克隆進行測序,實驗共得到成功測序的克隆1087個。用NCBI網(wǎng)站的BLAST功能進行數(shù)據(jù)庫檢索,得到符合ORFs讀框的克隆833個。對應(yīng)于非冗余獵物分子185個,鼠疫菌誘餌蛋白92個,及由此構(gòu)成的359個相互作用對,組成我們的核心數(shù)據(jù)集。實驗未篩選到文獻報道的已知相互作用。在這359對相互作用對中,60對出現(xiàn)頻率為兩次,91對出現(xiàn)頻率在三次或三次以上,分別占總數(shù)的16.7%和25.5%,說明我們的篩選結(jié)果可信度較高。 隨后采用實驗驗證和生物信息學(xué)分析的方法進一步評價相互作用的可信度。實驗部分,進行了酵母回轉(zhuǎn)實驗和GST pull down實驗。我們對讀框正確的359對相互作用進行了回轉(zhuǎn)實驗,經(jīng)過表型鑒定,208對相互作用表現(xiàn)為陽性,陽性率為57.9%。得到對應(yīng)于67個誘餌、109個獵物蛋白的208對相互作用。挑選酵母回轉(zhuǎn)實驗陽性的鼠疫菌和人蛋白相互對21個,進行GST pull down實驗驗證,約占全部回轉(zhuǎn)陽性的相互作用對的10%。21對相互作用中涉及6個獵物蛋白,由于其中1個獵物蛋白不能釋放到上清中,導(dǎo)致7對相互作用暫時無法驗證。在剩余的14對相互作用中,13對驗證為陽性,陽性率為92.8%,證明通過Y2H篩選及回轉(zhuǎn)驗證實驗得到的208對鼠疫菌和人蛋白之間的相互作用數(shù)據(jù)可靠性較高。利用雙雜交篩選獲得數(shù)據(jù),參考文獻及充分挖掘現(xiàn)有的公共數(shù)據(jù)庫(BIND,BioGRID,DIP,GeneRIF,HPRD,IntAct,MINT和Reactome等),使用Cytoscape軟件繪制并分析網(wǎng)絡(luò)的結(jié)構(gòu)和功能特點,評價網(wǎng)絡(luò)的可靠性。從文獻中的人PPI網(wǎng)絡(luò)中提取與鼠疫菌蛋白發(fā)生相互作用的人的蛋白(HYT, human protein targeted by Y.pestis proteins)及這些蛋白間的相互作用,得到了HYT-HYT之間相互作用的網(wǎng)絡(luò)。將此網(wǎng)絡(luò)與鼠疫菌蛋白與人蛋白之間的相互作用網(wǎng)絡(luò)融合得到了一個有176個節(jié)點,252條連線的網(wǎng)絡(luò)。代表包括67個鼠疫菌蛋白和109個人蛋白的252對相互作用,其中含有109個HYT在已知的人PPI網(wǎng)絡(luò)中拓展得到的44對相互作用。文獻中報道的112個與EB病毒相互作用的人蛋白中有26個(26/112=23%)也與鼠疫菌相互作用,提示病毒與細菌可能在攻擊宿主防御系統(tǒng)時采用了相似的策略,都是攻擊人體PPI網(wǎng)絡(luò)中的重要節(jié)點。這與目前已有的病原與宿主PPI的研究結(jié)果一致。在用GO biological process ID進行富集分析時發(fā)現(xiàn),參與物種間相互作用的蛋白、多物種相互作用的蛋白、細胞黏附蛋白和一些細胞內(nèi)的負調(diào)控蛋白得到顯著性的富集;在用GO molecular function ID進行富集分析時發(fā)現(xiàn),細胞骨架蛋白、轉(zhuǎn)錄因子結(jié)合蛋白、整合素蛋白、蛋白酶體蛋白等得到顯著性的富集。這些蛋白很可能在鼠疫菌致病過程中發(fā)揮重要作用。說明我們篩到的PPI可能與鼠疫菌的感染過程密切相關(guān)。最后,我們根據(jù)深入文獻調(diào)研,對篩選到的潛在相互作用進行了推測性的分析。 鼠疫菌和宿主間的相互作用就是一系列鼠疫菌與人體蛋白的相互作用產(chǎn)成的協(xié)同作用,我們篩到的PPI在鼠疫菌感染人體的過程中具體發(fā)揮怎樣的作用還有待于進一步深入的研究。本文以高通量的篩選方法研究鼠疫菌與人體蛋白之間的相互作用,獲得了相互作用的初步網(wǎng)絡(luò),對網(wǎng)絡(luò)進行了拓撲結(jié)構(gòu)和功能分析,揭示了鼠疫菌與人之間PPI的主要方式,為深入研究鼠疫菌的致病機制提供了靶標。 第二部分:鼠疫菌T3SS內(nèi)PPI的研究 pCD1質(zhì)粒為3種對人致病的耶爾森氏菌所共有的,是耶爾森氏菌必須的毒力因子,它編碼T3SS。該系統(tǒng)在鼠疫菌表面形成針狀小體,能將至少6種效應(yīng)蛋白(Yersinia outer membrane proteins,Yops)注射到宿主細胞中去。Yops一般具有蛋白酶的活性,通過對宿主蛋白的作用改變宿主細胞骨架結(jié)構(gòu)、抵抗吞噬作用、干擾信號轉(zhuǎn)導(dǎo)通路,從而抑制宿主正常的免疫響應(yīng)。 證實T3SS內(nèi)部不同組分之間的PPI有助于預(yù)測和闡明Ysc機制的結(jié)構(gòu)和功能。本實驗選取鼠疫菌pCD1質(zhì)粒編碼基因中去除復(fù)制、分配相關(guān)基因、轉(zhuǎn)座酶基因等后剩余57個基因,應(yīng)用Y2H陣列篩選技術(shù),進行T3SS內(nèi)部相互作用的研究。實驗共獲得19對相互作用蛋白,其中的8對是以前的研究中曾經(jīng)報道過的,剩余11對未見描述。相互作用主要分為三類:伴侶分子和分泌底物的相互作用,分泌調(diào)控復(fù)合體的相互作用與其他相互作用。有些相互作用發(fā)生在未知功能的假定蛋白之間;有些假定蛋白與已知的T3SS成分相互作用,提示他們可能是Ysc分泌機制新的成員。這些實驗結(jié)果將有助于將來進一步闡明T3SS的結(jié)構(gòu)和功能。確切的相互作用及意義有待于進一步研究。 第三部分:負調(diào)控因子LcrG對T3SS基因表達影響的研究 耶爾森氏菌T3SS由溫度、鈣離子、谷氨酸水平、PH值、營養(yǎng)獲得和接觸真核細胞等環(huán)境因素緊密調(diào)節(jié),并與病原體的生命周期密切聯(lián)系。T3SS裝置又稱作低鈣反應(yīng)刺激元(LCRS)。已知LcrG是Yops分泌的負調(diào)控因子,在37℃,體外Ca2+存在/缺失的條件下,lcrG突變株持續(xù)分泌Yops,并伴隨生長抑制。 為了更好的了解LcrG在調(diào)控鼠疫菌LCRS中的作用,我們構(gòu)建了一個非極性的lcrG突變株,進行了比較轉(zhuǎn)錄組學(xué)分析。實驗發(fā)現(xiàn),lcrG突變株相比野生株有很多T3SS基因上調(diào)。免疫印記實驗分析同樣表明lcrG突變株分泌更多的YopM和LcrV蛋白。此外,通過體外(HeLa細胞和巨噬細胞)和體內(nèi)(小鼠全身感染)實驗分析lcrG突變株,均顯示毒力顯著減弱:在巨噬細胞中阻止TNF-α分泌的能力嚴重受損;BALB/c小鼠的LD50增加約600倍。盡管Yops在突變株中過表達,但轉(zhuǎn)位到真核細胞中卻明顯受到阻礙,這一結(jié)論以前的報道一致。因此我們推斷,LcrG不僅負調(diào)控Yops分泌,而且可能也負調(diào)控T3SS基因轉(zhuǎn)錄,使Yop合成和分泌保持在一個合適的水平。這一功能可能是通過間接機制顯示的。
[Abstract]:The plague is a strong infectious disease caused by the plague Jerson Prand, and there have been three world pandemics in history. The pathogenic mechanism of Yersinia pestis depends on the interaction between it and the host. It reveals that the protein interaction between the Yersinia pestis and the human is the important basis for understanding its pathogenesis. In order to provide clues for the study of effective measures to prevent and control the occurrence and transmission of plague, this study includes three parts: first, high throughput yeast two hybrid (Y2H) is used to screen the human spleen cDNA library by using the speculating virulence related protein of Yersinia pestis as bait. The aim of this study is to obtain the preliminary network of protein interaction between Yersinia and human. And a number of important protein interactions (PPI) have been obtained to provide a target for further study of the pathogenic mechanism of Yersinia pestis; secondly, Y2H array screening technique is used to screen the interaction between protein in the III secretory system (T3SS) of Yersinia pestis (T3SS), which lays the foundation for further clarifying the structure and function of T3SS; third, to further understand the negative effect. The role of regulatory factor LcrG in the expression of T3SS gene in Yersinia pestis was studied by comparative transcriptional spectroscopy of the wild strain of Yersinia pestis and lcrG mutant by whole genome chip.
Part one: the interaction between Yersinia pestis virulence associated proteins and human proteins.
According to the results of comparative genomics of Yersinia pestis and Mycobacterium tuberculosis, and the results of the expression profiles of Yersinia pestis under different stimuli, serum antibody spectrum, and protein chip screening test results, the latest progress in the related research of Yersinia pestis in reference literature finally determines the egg Yersinia pestis eggs that may interact with human protein. White 152 (3.75% of the whole genome of Yersinia pestis). Using high throughput Y2H technology to screen the PPI. between Yersinia pestis and human, ProQuest? Two-Hybrid System based on Gateway recombination technology was used to facilitate high throughput parallel operation. Through Gateway recombination technology, 154 bait carriers were successfully constructed, corresponding to the OR of 152 Yersinia pestis protein. Fs. there are 3 self activated bait: YPCD1.26c, YPCD1.31c and YPMT1.61c..
At least one human spleen cDNA library was screened by large-scale Y2H screening by the sequence transformation method. The candidate clones on the plate were screened by the growth experiment to identify the phenotype of HIS and ADE, and the lacZ reporter gene phenotypes were identified by X-Gal analysis. The positive clones were sequenced. A total of 1087 clones were sequenced successfully. With the BLAST function of the NCBI website, the database was retrieved, and 833 clones consistent with the ORFs reading frame were obtained. It corresponds to 185 non redundant prey molecules, 92 bait protein of Yersinia pestis, and the resulting 359 interaction pairs, which constitute our core data set. In the 359 pairs of interaction pairs, the frequency of 60 pairs of pairs was two times, and the frequency of 91 pairs was three or more, accounting for 16.7% and 25.5% of the total, indicating that the results of our screening were more reliable.
Then experimental validation and bioinformatics analysis were used to further evaluate the reliability of interaction. In the experimental section, the yeast rotation experiment and the GST pull down experiment were carried out. We carried out a rotary experiment on the correct 359 pairs of interaction of the reading frame. After the phenotypic identification, 208 pairs of interactions were positive and the positive rate was 57.9%.. To the interaction between 67 bait and 109 prey proteins, 21 of Yersinia pestis and human proteins were selected for yeast gyration test positive, and 21 were tested by GST pull down, which accounted for 6 hunting proteins in the interaction of all positive interaction pairs, because 1 of them could not be released. In the supernatant, 7 pairs of interactions could not be verified for the time being. In the remaining 14 pairs of interaction, 13 pairs of tests were positive and the positive rate was 92.8%. It proved that the data of the interaction between the 208 pairs of Yersinia and human protein obtained through Y2H screening and rotary validation experiment were higher. Mining existing public databases (BIND, BioGRID, DIP, GeneRIF, HPRD, IntAct, MINT and Reactome, etc.), using Cytoscape software to draw and analyze the structure and function characteristics of the network and evaluate the reliability of the network. By Y.pestis proteins and the interaction between these proteins, the interaction network between HYT-HYT and the interaction network between the protein and human protein of Yersinia pestis was fused to obtain a network of 176 nodes and 252 connections, representing the 252 pairs of interactions including 67 pestis protein and 109 individual proteins. 109 of them have 44 pairs of HYT interactions in the known human PPI network. 112 of the human proteins that interact with EB viruses in the literature have 26 (26/112=23%) interacting with Yersinia, suggesting that the virus and bacteria may have adopted a similar strategy when attacking the host defense system, both of which attack the human PPI network. An important node in the collaterals. This is in accordance with the current research results of the existing pathogen and host PPI. In the enrichment analysis of GO biological process ID, it was found that proteins interacting with other species, proteins interacting with multiple species, cell adhesion proteins and negative regulatory proteins in some cells were enriched, and GO Mo was used. The enrichment analysis of lecular function ID found that cytoskeleton protein, transcription factor binding protein, integrin protein, proteasome protein and so on were significantly enriched. These proteins are likely to play an important role in the pathogenesis of Yersinia pestis. It shows that the PPI we sifted can be closely related to the infection process of Yersinia pestis. Finally, We conducted a speculative analysis of the potential interactions screened based on in-depth literature research.
The interaction between the Yersinia pestis and the host is a series of synergistic effects of the interaction of Yersinia pestis and human proteins. The specific role of the PPI we sifted in the process of Yersinia pestis infection is still to be further studied. This paper studies the relationship between Yersinia pestis and human proteins by high flux screening. The interaction of the interaction, obtained the interaction of the primary network, the network topology and function analysis, reveal the main way of PPI between Yersinia pestis and human, providing a target for the in-depth study of the pathogenic mechanism of Yersinia pestis.
The second part: the study of Yersinia pestis T3SS PPI
The pCD1 plasmid, which is common to 3 human pathogenic Yersinia bacteria, is a virulence factor that Jerson Prand must. It encodes a T3SS. system to form a needle like body on the surface of Yersinia pestis, and can inject at least 6 Effect proteins (Yersinia outer membrane proteins, Yops) into the host cells to have the activity of protease and through the action of the protease. The role of host protein changes the host cytoskeleton structure, resists phagocytosis, interferes with signal transduction pathways, and inhibits the host's normal immune response.
It was confirmed that the PPI between different components of T3SS was helpful to predict and clarify the structure and function of the Ysc mechanism. In this experiment, we selected the pCD1 plasmid encoding gene of Yersinia pestis to remove the replicas, distribute the related genes, the transposase gene and other 57 genes, and applied the Y2H array screening technique to study the internal interaction of T3SS. The experiment obtained 19 pairs of experiments. The 8 pairs of interacting proteins, which were previously reported in previous studies, have not been described in the remaining 11 pairs. Interaction is mainly divided into three categories: interaction between chaperones and secretory substrates, secretion of regulatory complexes and other interactions. Some interactions occur between the presumed proteins of unknown functions; some are false. The interaction between fixed protein and known T3SS components suggests that they may be new members of the Ysc secretory mechanism. These results will help to further clarify the structure and function of T3SS in the future. The exact interaction and significance need to be further studied.
The third part: the effect of negative regulatory factor LcrG on T3SS gene expression.
The T3SS T3SS is closely regulated by the environmental factors such as temperature, calcium ion, glutamic acid level, pH, nutrient acquisition and contact eukaryotic cells, and closely related to the life cycle of the pathogens called the.T3SS device called the low calcium response stimulant (LCRS). The known LcrG is a negative regulator of Yops secretion, under the condition of Ca2+ presence / absence in vitro, at 37. The lcrG mutant continued to secrete Yops, accompanied by growth inhibition.
In order to better understand the role of LcrG in the regulation of the LCRS of Yersinia pestis, we constructed a non polar lcrG mutant and carried out a comparative transcriptional analysis. It was found that the lcrG mutant had a lot of T3SS gene up regulation compared to the wild strain. The immunoimprint analysis also showed that the lcrG mutant secreted more YopM and LcrV protein. The analysis of lcrG mutants in vitro (HeLa cells and macrophages) and in vivo (murine systemic infection) showed that the virulence was significantly weakened: the ability to prevent TNF- alpha secretion in macrophages was severely impaired; the LD50 of the BALB/c mice increased by about 600 times. Although Yops was overexpressed in the mutant strain, the transposition to eukaryotic cells was significantly hindered. This conclusion is consistent with previous reports. Therefore, we infer that LcrG not only negatively regulates the secretion of Yops, but also may negatively regulate T3SS gene transcription, making Yop synthesis and secretion at a suitable level. This function may be shown by indirect mechanism.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R378
[Abstract]:The plague is a strong infectious disease caused by the plague Jerson Prand, and there have been three world pandemics in history. The pathogenic mechanism of Yersinia pestis depends on the interaction between it and the host. It reveals that the protein interaction between the Yersinia pestis and the human is the important basis for understanding its pathogenesis. In order to provide clues for the study of effective measures to prevent and control the occurrence and transmission of plague, this study includes three parts: first, high throughput yeast two hybrid (Y2H) is used to screen the human spleen cDNA library by using the speculating virulence related protein of Yersinia pestis as bait. The aim of this study is to obtain the preliminary network of protein interaction between Yersinia and human. And a number of important protein interactions (PPI) have been obtained to provide a target for further study of the pathogenic mechanism of Yersinia pestis; secondly, Y2H array screening technique is used to screen the interaction between protein in the III secretory system (T3SS) of Yersinia pestis (T3SS), which lays the foundation for further clarifying the structure and function of T3SS; third, to further understand the negative effect. The role of regulatory factor LcrG in the expression of T3SS gene in Yersinia pestis was studied by comparative transcriptional spectroscopy of the wild strain of Yersinia pestis and lcrG mutant by whole genome chip.
Part one: the interaction between Yersinia pestis virulence associated proteins and human proteins.
According to the results of comparative genomics of Yersinia pestis and Mycobacterium tuberculosis, and the results of the expression profiles of Yersinia pestis under different stimuli, serum antibody spectrum, and protein chip screening test results, the latest progress in the related research of Yersinia pestis in reference literature finally determines the egg Yersinia pestis eggs that may interact with human protein. White 152 (3.75% of the whole genome of Yersinia pestis). Using high throughput Y2H technology to screen the PPI. between Yersinia pestis and human, ProQuest? Two-Hybrid System based on Gateway recombination technology was used to facilitate high throughput parallel operation. Through Gateway recombination technology, 154 bait carriers were successfully constructed, corresponding to the OR of 152 Yersinia pestis protein. Fs. there are 3 self activated bait: YPCD1.26c, YPCD1.31c and YPMT1.61c..
At least one human spleen cDNA library was screened by large-scale Y2H screening by the sequence transformation method. The candidate clones on the plate were screened by the growth experiment to identify the phenotype of HIS and ADE, and the lacZ reporter gene phenotypes were identified by X-Gal analysis. The positive clones were sequenced. A total of 1087 clones were sequenced successfully. With the BLAST function of the NCBI website, the database was retrieved, and 833 clones consistent with the ORFs reading frame were obtained. It corresponds to 185 non redundant prey molecules, 92 bait protein of Yersinia pestis, and the resulting 359 interaction pairs, which constitute our core data set. In the 359 pairs of interaction pairs, the frequency of 60 pairs of pairs was two times, and the frequency of 91 pairs was three or more, accounting for 16.7% and 25.5% of the total, indicating that the results of our screening were more reliable.
Then experimental validation and bioinformatics analysis were used to further evaluate the reliability of interaction. In the experimental section, the yeast rotation experiment and the GST pull down experiment were carried out. We carried out a rotary experiment on the correct 359 pairs of interaction of the reading frame. After the phenotypic identification, 208 pairs of interactions were positive and the positive rate was 57.9%.. To the interaction between 67 bait and 109 prey proteins, 21 of Yersinia pestis and human proteins were selected for yeast gyration test positive, and 21 were tested by GST pull down, which accounted for 6 hunting proteins in the interaction of all positive interaction pairs, because 1 of them could not be released. In the supernatant, 7 pairs of interactions could not be verified for the time being. In the remaining 14 pairs of interaction, 13 pairs of tests were positive and the positive rate was 92.8%. It proved that the data of the interaction between the 208 pairs of Yersinia and human protein obtained through Y2H screening and rotary validation experiment were higher. Mining existing public databases (BIND, BioGRID, DIP, GeneRIF, HPRD, IntAct, MINT and Reactome, etc.), using Cytoscape software to draw and analyze the structure and function characteristics of the network and evaluate the reliability of the network. By Y.pestis proteins and the interaction between these proteins, the interaction network between HYT-HYT and the interaction network between the protein and human protein of Yersinia pestis was fused to obtain a network of 176 nodes and 252 connections, representing the 252 pairs of interactions including 67 pestis protein and 109 individual proteins. 109 of them have 44 pairs of HYT interactions in the known human PPI network. 112 of the human proteins that interact with EB viruses in the literature have 26 (26/112=23%) interacting with Yersinia, suggesting that the virus and bacteria may have adopted a similar strategy when attacking the host defense system, both of which attack the human PPI network. An important node in the collaterals. This is in accordance with the current research results of the existing pathogen and host PPI. In the enrichment analysis of GO biological process ID, it was found that proteins interacting with other species, proteins interacting with multiple species, cell adhesion proteins and negative regulatory proteins in some cells were enriched, and GO Mo was used. The enrichment analysis of lecular function ID found that cytoskeleton protein, transcription factor binding protein, integrin protein, proteasome protein and so on were significantly enriched. These proteins are likely to play an important role in the pathogenesis of Yersinia pestis. It shows that the PPI we sifted can be closely related to the infection process of Yersinia pestis. Finally, We conducted a speculative analysis of the potential interactions screened based on in-depth literature research.
The interaction between the Yersinia pestis and the host is a series of synergistic effects of the interaction of Yersinia pestis and human proteins. The specific role of the PPI we sifted in the process of Yersinia pestis infection is still to be further studied. This paper studies the relationship between Yersinia pestis and human proteins by high flux screening. The interaction of the interaction, obtained the interaction of the primary network, the network topology and function analysis, reveal the main way of PPI between Yersinia pestis and human, providing a target for the in-depth study of the pathogenic mechanism of Yersinia pestis.
The second part: the study of Yersinia pestis T3SS PPI
The pCD1 plasmid, which is common to 3 human pathogenic Yersinia bacteria, is a virulence factor that Jerson Prand must. It encodes a T3SS. system to form a needle like body on the surface of Yersinia pestis, and can inject at least 6 Effect proteins (Yersinia outer membrane proteins, Yops) into the host cells to have the activity of protease and through the action of the protease. The role of host protein changes the host cytoskeleton structure, resists phagocytosis, interferes with signal transduction pathways, and inhibits the host's normal immune response.
It was confirmed that the PPI between different components of T3SS was helpful to predict and clarify the structure and function of the Ysc mechanism. In this experiment, we selected the pCD1 plasmid encoding gene of Yersinia pestis to remove the replicas, distribute the related genes, the transposase gene and other 57 genes, and applied the Y2H array screening technique to study the internal interaction of T3SS. The experiment obtained 19 pairs of experiments. The 8 pairs of interacting proteins, which were previously reported in previous studies, have not been described in the remaining 11 pairs. Interaction is mainly divided into three categories: interaction between chaperones and secretory substrates, secretion of regulatory complexes and other interactions. Some interactions occur between the presumed proteins of unknown functions; some are false. The interaction between fixed protein and known T3SS components suggests that they may be new members of the Ysc secretory mechanism. These results will help to further clarify the structure and function of T3SS in the future. The exact interaction and significance need to be further studied.
The third part: the effect of negative regulatory factor LcrG on T3SS gene expression.
The T3SS T3SS is closely regulated by the environmental factors such as temperature, calcium ion, glutamic acid level, pH, nutrient acquisition and contact eukaryotic cells, and closely related to the life cycle of the pathogens called the.T3SS device called the low calcium response stimulant (LCRS). The known LcrG is a negative regulator of Yops secretion, under the condition of Ca2+ presence / absence in vitro, at 37. The lcrG mutant continued to secrete Yops, accompanied by growth inhibition.
In order to better understand the role of LcrG in the regulation of the LCRS of Yersinia pestis, we constructed a non polar lcrG mutant and carried out a comparative transcriptional analysis. It was found that the lcrG mutant had a lot of T3SS gene up regulation compared to the wild strain. The immunoimprint analysis also showed that the lcrG mutant secreted more YopM and LcrV protein. The analysis of lcrG mutants in vitro (HeLa cells and macrophages) and in vivo (murine systemic infection) showed that the virulence was significantly weakened: the ability to prevent TNF- alpha secretion in macrophages was severely impaired; the LD50 of the BALB/c mice increased by about 600 times. Although Yops was overexpressed in the mutant strain, the transposition to eukaryotic cells was significantly hindered. This conclusion is consistent with previous reports. Therefore, we infer that LcrG not only negatively regulates the secretion of Yops, but also may negatively regulate T3SS gene transcription, making Yop synthesis and secretion at a suitable level. This function may be shown by indirect mechanism.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R378
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1 李鐵強;吳永英;范洪臣;王立群;;酵母雙雜交系統(tǒng)研究進展[J];生物信息學(xué);2008年01期
2 孫玉寧,李春榮,靳更林;酵母質(zhì)粒PCR擴增及酶切在酵母雙雜交中的運用[J];承德醫(yī)學(xué)院學(xué)報;2005年03期
3 劉卉;潘欣;李慧艷;張佩景;李衛(wèi)華;張學(xué)敏;周濤;;應(yīng)用酵母雙雜交篩選與CUEDC2相互作用的蛋白質(zhì)[J];生物技術(shù)通訊;2007年05期
4 武君;沈秀張;林吉進;;蛋白質(zhì)與蛋白質(zhì)相互作用研究技術(shù)[J];中國分子心臟病學(xué)雜志;2008年02期
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