噬菌体PaP3与宿主基因组水平的相互作用及抑菌基因orf70.1的功能鉴定
发布时间：2018-08-08 17:05
【摘要】：铜绿假单胞菌(PseudomonasaP3aeruginosa)是一种人类条件致病性的革兰氏阴性菌,该菌有非常强的适应能力,在自然界中广泛存在。铜绿假单胞菌的感染常见于呼吸道,烧伤和创伤等,是导致院内感染的主要病原体之一。近年来,由于抗生素的滥用,加速了耐药菌的出现和扩散,使现有的抗生素失效。耐药菌对人类的威胁成为全球瞩目的健康问题,有研究将导致院内感染最常见的六种多重耐药细菌称为“ESKAPE”(EnterococcusaP3faecium,StaphylococcusaP3aureus,KlebsiellaaP3pneumoniae,AcinetobacteraP3baumanii,PseudomonasaP3aeruginosa,andaP3EnterobacteraP3species),这些细菌耐药情况非常严重,有的甚至是全耐药的。铜绿假单胞菌的基因组是最大的细菌基因组之一,含有多种药物抗性基因。由于新的抗生素的研究发展远远滞后于细菌的突变速度,这使得多重耐药的铜绿假单胞菌感染治疗相当困难。自从1915年首次发现噬菌体后不久,人们就成功地将噬菌体应用到铜绿假单胞菌感染的治疗中,并取得了很好的效果。自然界中,作为细菌的天敌,噬菌体的数目是细菌的十倍,是一个巨大的抗菌制剂的资源池。然而,目前已测序的噬菌体只有一千多种,人们对噬菌体-宿主之间的相互作用的了解更是冰山一角。这些知识的匮乏,阻碍了人们对噬菌体这一资源的利用。因此,为了充分利用噬菌体来对抗目前形势严峻的耐药细菌,我们迫切地需要对噬菌体-宿主相互作用及噬菌体基因功能有更深入地了解。本文根据噬菌体的一步生长曲线,利用表达谱芯片技术考察了铜绿假单胞菌噬菌体PaP3与其宿主-铜绿假单胞菌PA3在不同时期的全基因组表达情况,并绘制了噬菌体-宿主基因之间的相互作用网络。以此为基础并结合前期工作,我们从噬菌体PaP3早期基因中筛选到一个具有抑菌活性的基因—orf70.1。接着通过包括GSTaP3pullaP3down表达谱芯片技术,RT-qaP3PCR分析,核磁共振(NMR)质谱和表型分析等技术,我们对orf70.1的功能进行了综合的分析。以下为本文的主要研究内容及结果:铜绿假单胞菌噬菌体PaP3对宿主菌全基因组基因表达的影响:我们根据噬菌体PaP3的一步生长曲线,选择了覆盖整个噬菌体生长时期的五个时间点,利用表达谱芯片技术考察了噬菌体PaP3对宿主铜绿假单胞菌PA3的全基因组表达的影响。结果显示,噬菌体PaP3感染宿主最严重的时期为感染中期(10-30aP3min),能导致38%的宿主基因出现差异表达,其中98%的差异表达基因为下调表达基因。这些差异表达基因涉及了宿主细菌非常广泛的功能以及具有时间依赖性的代谢途径。铜绿假单胞菌噬菌体PaP3与宿主基因间相互作用网络的构建:根据芯片结果,我们利用Pearson相关系数计算了噬菌体和宿主基因之间的共表达关系,确定了噬菌体PaP3基因与宿主转录调控因子之间的相互作用,绘制了噬菌体PaP3感染早期,中期,晚期以及整个感染时期噬菌体-宿主的相互作用网络图。通过上述分析我们得到以下结论:1)噬菌体PaP3基因与宿主转录调控因子之间存在明显的负相关关系,说明噬菌体可能通过对宿主转录调控因子的抑制作用导致宿主的基因出现大规模下调表达;2)由于在同一个共表达网络中的基因可能具有相似的功能,通过亚网络的基因聚类,我们推测出几个噬菌体未知基因可能的生物学功能;3)宿主的氨基酸代谢和小分子转运功能相关基因是最容易受到噬菌体调控的基因。噬菌体PaP3基因的重新注释:综合利用六个基因预测平台对噬菌体PaP3的基因进行重新预测分析,并利用RNA-seq对预测出来的基因进行表达验证。最终将噬菌体PaP3的基因由原来预测的71个基因补充至126个基因。抑菌基因orf70.1及其靶蛋白的鉴定:根据前期工作,我们从两个能与宿主蛋白相互作用的噬菌体蛋白中,鉴定出一个能够编码抑菌蛋白的基因—orf70.1,其编码产物命名为gp70.1(geneaP3product,gp)。在gp70.1表达的情况下,宿主菌PA3在固体培养基上呈现针尖样小菌落,在液体培养基中生长周期被延迟。生物信息分析和BLASTP显示,gp70.1是一个新的未知功能的蛋白,预测小大为13kd左右,不存在保守结构域,在已知的蛋白数据库中没有检测到有意义的同源蛋白。我们纯化了gp70.1,其大小与生物信息预测相符。此外我们还制备了gp70.1的多克隆抗体,利用GST-pullaP3down实验,证实了gp70.1能在体外与RpoaP3S直接结合。Gp70.1对宿主菌RpoaP3S功能的影响:RpoaP3S是铜绿假单胞菌中重要的应激条件全局性转录调控因子,分析结果显示,RpoaP3S与gp70.1的相互作用能抑制RpoaP3S调控的应激反应,生物膜形成以及毒力等。多种方法联用综合分析抑菌蛋白gp70.1对铜绿假单胞菌的影响:联合表达谱芯片技术,RT-qaP3PCR,基于核磁共振质谱(NMR)的代谢组分析以及一系列表型分析,我们综合考察了gp70.1对铜绿假单胞菌PA3的影响。结果显示,在细菌生长的延迟期,对数期和稳定期一共有178个宿主菌基因受到gp70.1的影响,这些差异表达的基因主要涉及细菌的细胞外功能和代谢功能。接着,NMR分析发现gp70.1对细菌的氨基酸代谢具有显著的抑制作用。表型分析显示,gp70.1对PA3的基本形态没有影响,但是能强烈地抑制细菌的胞外蛋白酶,胞外多糖,胞外纤维素酶,绿脓色素以及运动功能。综上所述,我们对铜绿假单胞菌噬菌体PaP3对宿主菌PA3全基因组转录的影响有了深入的了解,并绘制的基因水平的噬菌体PaP3-宿主相互作用网络,为后续的噬菌体抗菌蛋白的筛选以及噬菌体-宿主相互作用的深入解析提供了研究基础。此外我们对噬菌体PaP3基因的补充注释,并在此基础上鉴定了一个新的噬菌体PaP3编码的抑菌蛋白及其靶蛋白。
[Abstract]:Pseudomonas aeruginosa (PseudomonasaP3aeruginosa) is a pathogenic gram negative bacteria of human condition. It has a very strong adaptability and exists widely in nature. The infection of Pseudomonas aeruginosa is common in respiratory tract, burns and wounds. It is one of the main pathogens causing nosocomial infection. In recent years, due to antibiotics Abuse has accelerated the emergence and diffusion of drug-resistant bacteria and invalidated existing antibiotics. The threat of resistant bacteria to human beings has become a global health problem. There are six types of multidrug resistant bacteria, known as the most common nosocomial infection, called "ESKAPE" (EnterococcusaP3faecium, StaphylococcusaP3aureus, KlebsiellaaP3pneumoniae, Acinetobac). TeraP3baumanii, PseudomonasaP3aeruginosa, andaP3EnterobacteraP3species), the bacterial resistance of these bacteria is very serious, and some are all drug-resistant. The genome of Pseudomonas aeruginosa is one of the largest bacterial genomes, and contains a variety of drug resistance genes. The development of the new antibiotic is lagging far behind the strain of bacteria. Degree, which makes the multidrug-resistant Pseudomonas aeruginosa infection very difficult. Since the first discovery of phage in 1915, the phage has been successfully applied to the treatment of Pseudomonas aeruginosa infection and has achieved good results. In nature, the number of bacteriophages, as the natural enemy of bacteria, is ten times that of bacteria. A huge pool of antimicrobial agents. However, there are only one thousand bacteriophages that have been sequenced. The understanding of the interaction between phage hosts is a corner of the iceberg. The lack of knowledge has hindered the use of the phage resources. Therefore, the full use of phages to combat the current situation is severe. In this paper, the expression of Pseudomonas aeruginosa PaP3 and Pseudomonas aeruginosa (Pseudomonas aeruginosa) (Pseudomonas aeruginosa) PA3 in the whole genome of Pseudomonas aeruginosa is investigated in this paper based on the one step growth curve of phage. The interaction network of phage host genes was plotted and based on the previous work, we screened a bacteriostat gene from the early phage PaP3 gene - orf70.1. and then through the GSTaP3pullaP3down expression spectrum chip technology, RT-qaP3PCR analysis, nuclear magnetic resonance (NMR) mass spectrometry and phenotypic scores. We analyzed the function of orf70.1 synthetically. The following is the main research content and the result: the effect of Pseudomonas aeruginosa bacteriophage PaP3 on the whole genome gene expression of host bacteria: we choose five time points of covering the whole period of phage growth according to the one step growth curve of phage PaP3. The effect of phage PaP3 on the whole genome expression of Pseudomonas aeruginosa PA3 was investigated by expression spectrum chip technology. The results showed that the most serious period of phage PaP3 infection was mid infection (10-30aP3min), which could lead to 38% differential expression of host genes, and 98% of the differentially expressed genes were down regulated genes. The different expression genes involved the very extensive function of the host bacteria and the time dependent metabolic pathway. The construction of the interaction network between Pseudomonas aeruginosa phage PaP3 and host gene: Based on the results of the chip, we used the Pearson correlation coefficient to calculate the co expression relationship between phage and host genes. The interaction between the PaP3 gene and the host transcriptional regulatory factor was used to draw a network map of phage PaP3 infection at early, middle, late, and entire infection period. The following conclusions were obtained: 1) there is a clear negative correlation between the phage PaP3 gene and the host transcriptional regulator. The relationship shows that phages may lead to a large-scale down-regulation of the host gene by inhibiting the host transcriptional regulation factor; 2) because genes in the same coexpression network may have similar functions, we conjecture the possible biological functions of several phage unknown genes by cluster of subnetwork genes; 3 The host's amino acid metabolism and small molecular transport related genes are the most susceptible genes regulated by phage. A new annotation of the phage PaP3 gene: a comprehensive use of six gene prediction platforms to repredict the gene of phage PaP3, and to verify the pretested genes by RNA-seq. The gene of the body PaP3 was supplemented by 71 genes predicted from the original to 126 genes. Identification of the bacteriostat gene orf70.1 and its target protein: according to the earlier work, we identified a gene orf70.1 that could encode bacteriostat protein from two phage proteins that interact with the host protein, and its encoding product was named gp70.1 (geneaP3produ CT, GP). In the case of gp70.1 expression, the host fungus PA3 presents a pinpoint like colony on the solid medium, and the growth cycle is delayed in the liquid medium. Bioinformatics analysis and BLASTP show that gp70.1 is a new unknown function protein, and the prediction is about 13kd, without a conservative domain, and not in the known protein database. We have detected a meaningful homologous protein. We purified gp70.1, the size of which coincide with the prediction of bioinformatics. In addition, we have prepared polyclonal antibodies of gp70.1. Using GST-pullaP3down experiments, we confirmed the effect of gp70.1 on RpoaP3S function of host bacteria directly associated with RpoaP3S in vitro with RpoaP3S: RpoaP3S is the medium weight of Pseudomonas aeruginosa. The results show that the interaction of RpoaP3S and gp70.1 can inhibit the stress response, biofilm formation and virulence of RpoaP3S regulated by the interaction of the stress conditions. A variety of methods combined to synthetically analyze the effect of bacteriostin gp70.1 on Pseudomonas aeruginosa: combined expression spectrum chip technology, RT-qaP3PCR, based on nuclear magnetic Co The metabolic group analysis of NMR and a series of phenotypic analysis were used to examine the effect of gp70.1 on Pseudomonas aeruginosa PA3. The results showed that in the delay period of bacterial growth, 178 host genes were affected by gp70.1 in the logarithmic and stable stages, and these differentially expressed genes were mainly related to the extracellular function of bacteria. And metabolic function. Then, NMR analysis found that gp70.1 had a significant inhibitory effect on the amino acid metabolism of bacteria. Phenotypic analysis showed that gp70.1 had no effect on the basic morphology of PA3, but could strongly inhibit the extracellular protease, extracellular polysaccharide, extracellular cellulase, green purulent pigments, and motor function. The effect of Pseudomonas sp. phage PaP3 on the whole genome transcript of host PA3 was deeply understood, and the gene level phage PaP3- host interaction network was drawn to provide the basis for the further analysis of the subsequent bacteriophage antibacterial proteins and the deep analysis of phage host interaction. In addition, we have been on the phage PaP3 base. A new bacteriophage PaP3 encoding bacteriostatic protein and its target protein was identified based on the supplement annotation.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378
，
本文编号：2172445
[Abstract]:Pseudomonas aeruginosa (PseudomonasaP3aeruginosa) is a pathogenic gram negative bacteria of human condition. It has a very strong adaptability and exists widely in nature. The infection of Pseudomonas aeruginosa is common in respiratory tract, burns and wounds. It is one of the main pathogens causing nosocomial infection. In recent years, due to antibiotics Abuse has accelerated the emergence and diffusion of drug-resistant bacteria and invalidated existing antibiotics. The threat of resistant bacteria to human beings has become a global health problem. There are six types of multidrug resistant bacteria, known as the most common nosocomial infection, called "ESKAPE" (EnterococcusaP3faecium, StaphylococcusaP3aureus, KlebsiellaaP3pneumoniae, Acinetobac). TeraP3baumanii, PseudomonasaP3aeruginosa, andaP3EnterobacteraP3species), the bacterial resistance of these bacteria is very serious, and some are all drug-resistant. The genome of Pseudomonas aeruginosa is one of the largest bacterial genomes, and contains a variety of drug resistance genes. The development of the new antibiotic is lagging far behind the strain of bacteria. Degree, which makes the multidrug-resistant Pseudomonas aeruginosa infection very difficult. Since the first discovery of phage in 1915, the phage has been successfully applied to the treatment of Pseudomonas aeruginosa infection and has achieved good results. In nature, the number of bacteriophages, as the natural enemy of bacteria, is ten times that of bacteria. A huge pool of antimicrobial agents. However, there are only one thousand bacteriophages that have been sequenced. The understanding of the interaction between phage hosts is a corner of the iceberg. The lack of knowledge has hindered the use of the phage resources. Therefore, the full use of phages to combat the current situation is severe. In this paper, the expression of Pseudomonas aeruginosa PaP3 and Pseudomonas aeruginosa (Pseudomonas aeruginosa) (Pseudomonas aeruginosa) PA3 in the whole genome of Pseudomonas aeruginosa is investigated in this paper based on the one step growth curve of phage. The interaction network of phage host genes was plotted and based on the previous work, we screened a bacteriostat gene from the early phage PaP3 gene - orf70.1. and then through the GSTaP3pullaP3down expression spectrum chip technology, RT-qaP3PCR analysis, nuclear magnetic resonance (NMR) mass spectrometry and phenotypic scores. We analyzed the function of orf70.1 synthetically. The following is the main research content and the result: the effect of Pseudomonas aeruginosa bacteriophage PaP3 on the whole genome gene expression of host bacteria: we choose five time points of covering the whole period of phage growth according to the one step growth curve of phage PaP3. The effect of phage PaP3 on the whole genome expression of Pseudomonas aeruginosa PA3 was investigated by expression spectrum chip technology. The results showed that the most serious period of phage PaP3 infection was mid infection (10-30aP3min), which could lead to 38% differential expression of host genes, and 98% of the differentially expressed genes were down regulated genes. The different expression genes involved the very extensive function of the host bacteria and the time dependent metabolic pathway. The construction of the interaction network between Pseudomonas aeruginosa phage PaP3 and host gene: Based on the results of the chip, we used the Pearson correlation coefficient to calculate the co expression relationship between phage and host genes. The interaction between the PaP3 gene and the host transcriptional regulatory factor was used to draw a network map of phage PaP3 infection at early, middle, late, and entire infection period. The following conclusions were obtained: 1) there is a clear negative correlation between the phage PaP3 gene and the host transcriptional regulator. The relationship shows that phages may lead to a large-scale down-regulation of the host gene by inhibiting the host transcriptional regulation factor; 2) because genes in the same coexpression network may have similar functions, we conjecture the possible biological functions of several phage unknown genes by cluster of subnetwork genes; 3 The host's amino acid metabolism and small molecular transport related genes are the most susceptible genes regulated by phage. A new annotation of the phage PaP3 gene: a comprehensive use of six gene prediction platforms to repredict the gene of phage PaP3, and to verify the pretested genes by RNA-seq. The gene of the body PaP3 was supplemented by 71 genes predicted from the original to 126 genes. Identification of the bacteriostat gene orf70.1 and its target protein: according to the earlier work, we identified a gene orf70.1 that could encode bacteriostat protein from two phage proteins that interact with the host protein, and its encoding product was named gp70.1 (geneaP3produ CT, GP). In the case of gp70.1 expression, the host fungus PA3 presents a pinpoint like colony on the solid medium, and the growth cycle is delayed in the liquid medium. Bioinformatics analysis and BLASTP show that gp70.1 is a new unknown function protein, and the prediction is about 13kd, without a conservative domain, and not in the known protein database. We have detected a meaningful homologous protein. We purified gp70.1, the size of which coincide with the prediction of bioinformatics. In addition, we have prepared polyclonal antibodies of gp70.1. Using GST-pullaP3down experiments, we confirmed the effect of gp70.1 on RpoaP3S function of host bacteria directly associated with RpoaP3S in vitro with RpoaP3S: RpoaP3S is the medium weight of Pseudomonas aeruginosa. The results show that the interaction of RpoaP3S and gp70.1 can inhibit the stress response, biofilm formation and virulence of RpoaP3S regulated by the interaction of the stress conditions. A variety of methods combined to synthetically analyze the effect of bacteriostin gp70.1 on Pseudomonas aeruginosa: combined expression spectrum chip technology, RT-qaP3PCR, based on nuclear magnetic Co The metabolic group analysis of NMR and a series of phenotypic analysis were used to examine the effect of gp70.1 on Pseudomonas aeruginosa PA3. The results showed that in the delay period of bacterial growth, 178 host genes were affected by gp70.1 in the logarithmic and stable stages, and these differentially expressed genes were mainly related to the extracellular function of bacteria. And metabolic function. Then, NMR analysis found that gp70.1 had a significant inhibitory effect on the amino acid metabolism of bacteria. Phenotypic analysis showed that gp70.1 had no effect on the basic morphology of PA3, but could strongly inhibit the extracellular protease, extracellular polysaccharide, extracellular cellulase, green purulent pigments, and motor function. The effect of Pseudomonas sp. phage PaP3 on the whole genome transcript of host PA3 was deeply understood, and the gene level phage PaP3- host interaction network was drawn to provide the basis for the further analysis of the subsequent bacteriophage antibacterial proteins and the deep analysis of phage host interaction. In addition, we have been on the phage PaP3 base. A new bacteriophage PaP3 encoding bacteriostatic protein and its target protein was identified based on the supplement annotation.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378
，
本文编号：2172445
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