一、鼠疫菌毒力相关蛋白与人蛋白相互作用的初步研究 二、鼠疫菌Ⅲ型分泌系统内蛋白相互作用及LcrG调控的研究
发布时间：2018-07-23 11:05
【摘要】： 鼠疫是由鼠疫耶尔森氏菌引发的的一种烈性传染病,历史上曾经发生三次人间鼠疫的世界大流行。鼠疫菌的致病机制依赖于它和宿主之间的相互作用而实现。揭示鼠疫菌与人之间的蛋白相互作用是深入了解其致病机制的重要基础,同时为研究有效的预防和控制鼠疫的发生和传播的手段提供线索。本研究包括三部分:首先,采用高通量的酵母双杂交技术(Y2H),以推测的鼠疫菌毒力相关蛋白为诱饵筛选人脾脏cDNA文库,旨在获得鼠疫菌与人之间蛋白相互作用的初步网络,并且获得一批重要的蛋白相互作用(PPI),为深入揭示鼠疫菌的致病机制提供研究靶标;其次,应用Y2H阵列筛选技术筛选鼠疫菌III型分泌系统(T3SS)内部蛋白质间的相互作用,为进一步阐明T3SS的结构和功能奠定基础;第三,为进一步了解负调控因子LcrG在鼠疫菌T3SS基因表达中的作用,应用全基因组芯片进行了鼠疫菌野生株和lcrG突变株的比较转录谱学研究。 第一部分:鼠疫菌毒力相关蛋白与人蛋白质相互作用的研究 我们根据鼠疫菌和假结核菌的比较基因组学研究结果和本实验室开展的鼠疫菌在不同刺激条件下的表达谱、血清抗体谱、蛋白芯片筛选的试验结果,参考文献中鼠疫菌相关研究的最新进展最终确定可能与人蛋白相互作用的鼠疫菌蛋白152个(占鼠疫菌整个基因组的3.75%)。利用高通量Y2H技术筛选鼠疫菌与人之间的PPI。本研究采用基于Gateway重组技术的ProQuest? Two-Hybrid System,便于高通量平行操作。通过Gateway重组技术,成功构建了154个诱饵载体,对应于152个鼠疫菌蛋白的ORFs。其中存在自激活的诱饵有3个,分别是:YPCD1.26c,YPCD1.31c和YPMT1.61c。 通过顺序转化法进行规模化Y2H筛选,共对151个无自激活活性的诱饵至少筛选一次人脾脏cDNA文库。筛选平板上长出的候选克隆分别通过生长实验鉴定HIS和ADE报告基因表型,X-Gal分析鉴定lacZ报告基因表型。对得到的阳性克隆进行测序,实验共得到成功测序的克隆1087个。用NCBI网站的BLAST功能进行数据库检索,得到符合ORFs读框的克隆833个。对应于非冗余猎物分子185个,鼠疫菌诱饵蛋白92个,及由此构成的359个相互作用对,组成我们的核心数据集。实验未筛选到文献报道的已知相互作用。在这359对相互作用对中,60对出现频率为两次,91对出现频率在三次或三次以上,分别占总数的16.7%和25.5%,说明我们的筛选结果可信度较高。 随后采用实验验证和生物信息学分析的方法进一步评价相互作用的可信度。实验部分,进行了酵母回转实验和GST pull down实验。我们对读框正确的359对相互作用进行了回转实验,经过表型鉴定,208对相互作用表现为阳性,阳性率为57.9%。得到对应于67个诱饵、109个猎物蛋白的208对相互作用。挑选酵母回转实验阳性的鼠疫菌和人蛋白相互对21个,进行GST pull down实验验证,约占全部回转阳性的相互作用对的10%。21对相互作用中涉及6个猎物蛋白,由于其中1个猎物蛋白不能释放到上清中,导致7对相互作用暂时无法验证。在剩余的14对相互作用中,13对验证为阳性,阳性率为92.8%,证明通过Y2H筛选及回转验证实验得到的208对鼠疫菌和人蛋白之间的相互作用数据可靠性较高。利用双杂交筛选获得数据,参考文献及充分挖掘现有的公共数据库(BIND,BioGRID,DIP,GeneRIF,HPRD,IntAct,MINT和Reactome等),使用Cytoscape软件绘制并分析网络的结构和功能特点,评价网络的可靠性。从文献中的人PPI网络中提取与鼠疫菌蛋白发生相互作用的人的蛋白(HYT, human protein targeted by Y.pestis proteins)及这些蛋白间的相互作用,得到了HYT-HYT之间相互作用的网络。将此网络与鼠疫菌蛋白与人蛋白之间的相互作用网络融合得到了一个有176个节点,252条连线的网络。代表包括67个鼠疫菌蛋白和109个人蛋白的252对相互作用,其中含有109个HYT在已知的人PPI网络中拓展得到的44对相互作用。文献中报道的112个与EB病毒相互作用的人蛋白中有26个(26/112=23%)也与鼠疫菌相互作用,提示病毒与细菌可能在攻击宿主防御系统时采用了相似的策略,都是攻击人体PPI网络中的重要节点。这与目前已有的病原与宿主PPI的研究结果一致。在用GO biological process ID进行富集分析时发现,参与物种间相互作用的蛋白、多物种相互作用的蛋白、细胞黏附蛋白和一些细胞内的负调控蛋白得到显著性的富集;在用GO molecular function ID进行富集分析时发现,细胞骨架蛋白、转录因子结合蛋白、整合素蛋白、蛋白酶体蛋白等得到显著性的富集。这些蛋白很可能在鼠疫菌致病过程中发挥重要作用。说明我们筛到的PPI可能与鼠疫菌的感染过程密切相关。最后,我们根据深入文献调研,对筛选到的潜在相互作用进行了推测性的分析。 鼠疫菌和宿主间的相互作用就是一系列鼠疫菌与人体蛋白的相互作用产成的协同作用,我们筛到的PPI在鼠疫菌感染人体的过程中具体发挥怎样的作用还有待于进一步深入的研究。本文以高通量的筛选方法研究鼠疫菌与人体蛋白之间的相互作用,获得了相互作用的初步网络,对网络进行了拓扑结构和功能分析,揭示了鼠疫菌与人之间PPI的主要方式,为深入研究鼠疫菌的致病机制提供了靶标。 第二部分:鼠疫菌T3SS内PPI的研究 pCD1质粒为3种对人致病的耶尔森氏菌所共有的,是耶尔森氏菌必须的毒力因子,它编码T3SS。该系统在鼠疫菌表面形成针状小体,能将至少6种效应蛋白(Yersinia outer membrane proteins,Yops)注射到宿主细胞中去。Yops一般具有蛋白酶的活性,通过对宿主蛋白的作用改变宿主细胞骨架结构、抵抗吞噬作用、干扰信号转导通路,从而抑制宿主正常的免疫响应。 证实T3SS内部不同组分之间的PPI有助于预测和阐明Ysc机制的结构和功能。本实验选取鼠疫菌pCD1质粒编码基因中去除复制、分配相关基因、转座酶基因等后剩余57个基因,应用Y2H阵列筛选技术,进行T3SS内部相互作用的研究。实验共获得19对相互作用蛋白,其中的8对是以前的研究中曾经报道过的,剩余11对未见描述。相互作用主要分为三类:伴侣分子和分泌底物的相互作用,分泌调控复合体的相互作用与其他相互作用。有些相互作用发生在未知功能的假定蛋白之间;有些假定蛋白与已知的T3SS成分相互作用,提示他们可能是Ysc分泌机制新的成员。这些实验结果将有助于将来进一步阐明T3SS的结构和功能。确切的相互作用及意义有待于进一步研究。 第三部分:负调控因子LcrG对T3SS基因表达影响的研究 耶尔森氏菌T3SS由温度、钙离子、谷氨酸水平、PH值、营养获得和接触真核细胞等环境因素紧密调节,并与病原体的生命周期密切联系。T3SS装置又称作低钙反应刺激元(LCRS)。已知LcrG是Yops分泌的负调控因子,在37℃,体外Ca2+存在/缺失的条件下,lcrG突变株持续分泌Yops,并伴随生长抑制。 为了更好的了解LcrG在调控鼠疫菌LCRS中的作用,我们构建了一个非极性的lcrG突变株,进行了比较转录组学分析。实验发现,lcrG突变株相比野生株有很多T3SS基因上调。免疫印记实验分析同样表明lcrG突变株分泌更多的YopM和LcrV蛋白。此外,通过体外(HeLa细胞和巨噬细胞)和体内(小鼠全身感染)实验分析lcrG突变株,均显示毒力显著减弱:在巨噬细胞中阻止TNF-α分泌的能力严重受损;BALB/c小鼠的LD50增加约600倍。尽管Yops在突变株中过表达,但转位到真核细胞中却明显受到阻碍,这一结论以前的报道一致。因此我们推断,LcrG不仅负调控Yops分泌,而且可能也负调控T3SS基因转录,使Yop合成和分泌保持在一个合适的水平。这一功能可能是通过间接机制显示的。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378
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