噬菌體PaP3與宿主基因組水平的相互作用及抑菌基因orf70.1的功能鑒定
發(fā)布時間：2018-08-08 17:05
【摘要】：銅綠假單胞菌(PseudomonasaP3aeruginosa)是一種人類條件致病性的革蘭氏陰性菌,該菌有非常強的適應(yīng)能力,在自然界中廣泛存在。銅綠假單胞菌的感染常見于呼吸道,燒傷和創(chuàng)傷等,是導(dǎo)致院內(nèi)感染的主要病原體之一。近年來,由于抗生素的濫用,加速了耐藥菌的出現(xiàn)和擴散,使現(xiàn)有的抗生素失效。耐藥菌對人類的威脅成為全球矚目的健康問題,有研究將導(dǎo)致院內(nèi)感染最常見的六種多重耐藥細(xì)菌稱為“ESKAPE”(EnterococcusaP3faecium,StaphylococcusaP3aureus,KlebsiellaaP3pneumoniae,AcinetobacteraP3baumanii,PseudomonasaP3aeruginosa,andaP3EnterobacteraP3species),這些細(xì)菌耐藥情況非常嚴(yán)重,有的甚至是全耐藥的。銅綠假單胞菌的基因組是最大的細(xì)菌基因組之一,含有多種藥物抗性基因。由于新的抗生素的研究發(fā)展遠(yuǎn)遠(yuǎn)滯后于細(xì)菌的突變速度,這使得多重耐藥的銅綠假單胞菌感染治療相當(dāng)困難。自從1915年首次發(fā)現(xiàn)噬菌體后不久,人們就成功地將噬菌體應(yīng)用到銅綠假單胞菌感染的治療中,并取得了很好的效果。自然界中,作為細(xì)菌的天敵,噬菌體的數(shù)目是細(xì)菌的十倍,是一個巨大的抗菌制劑的資源池。然而,目前已測序的噬菌體只有一千多種,人們對噬菌體-宿主之間的相互作用的了解更是冰山一角。這些知識的匱乏,阻礙了人們對噬菌體這一資源的利用。因此,為了充分利用噬菌體來對抗目前形勢嚴(yán)峻的耐藥細(xì)菌,我們迫切地需要對噬菌體-宿主相互作用及噬菌體基因功能有更深入地了解。本文根據(jù)噬菌體的一步生長曲線,利用表達(dá)譜芯片技術(shù)考察了銅綠假單胞菌噬菌體PaP3與其宿主-銅綠假單胞菌PA3在不同時期的全基因組表達(dá)情況,并繪制了噬菌體-宿主基因之間的相互作用網(wǎng)絡(luò)。以此為基礎(chǔ)并結(jié)合前期工作,我們從噬菌體PaP3早期基因中篩選到一個具有抑菌活性的基因—orf70.1。接著通過包括GSTaP3pullaP3down表達(dá)譜芯片技術(shù),RT-qaP3PCR分析,核磁共振(NMR)質(zhì)譜和表型分析等技術(shù),我們對orf70.1的功能進(jìn)行了綜合的分析。以下為本文的主要研究內(nèi)容及結(jié)果:銅綠假單胞菌噬菌體PaP3對宿主菌全基因組基因表達(dá)的影響:我們根據(jù)噬菌體PaP3的一步生長曲線,選擇了覆蓋整個噬菌體生長時期的五個時間點,利用表達(dá)譜芯片技術(shù)考察了噬菌體PaP3對宿主銅綠假單胞菌PA3的全基因組表達(dá)的影響。結(jié)果顯示,噬菌體PaP3感染宿主最嚴(yán)重的時期為感染中期(10-30aP3min),能導(dǎo)致38%的宿主基因出現(xiàn)差異表達(dá),其中98%的差異表達(dá)基因為下調(diào)表達(dá)基因。這些差異表達(dá)基因涉及了宿主細(xì)菌非常廣泛的功能以及具有時間依賴性的代謝途徑。銅綠假單胞菌噬菌體PaP3與宿主基因間相互作用網(wǎng)絡(luò)的構(gòu)建:根據(jù)芯片結(jié)果,我們利用Pearson相關(guān)系數(shù)計算了噬菌體和宿主基因之間的共表達(dá)關(guān)系,確定了噬菌體PaP3基因與宿主轉(zhuǎn)錄調(diào)控因子之間的相互作用,繪制了噬菌體PaP3感染早期,中期,晚期以及整個感染時期噬菌體-宿主的相互作用網(wǎng)絡(luò)圖。通過上述分析我們得到以下結(jié)論:1)噬菌體PaP3基因與宿主轉(zhuǎn)錄調(diào)控因子之間存在明顯的負(fù)相關(guān)關(guān)系,說明噬菌體可能通過對宿主轉(zhuǎn)錄調(diào)控因子的抑制作用導(dǎo)致宿主的基因出現(xiàn)大規(guī)模下調(diào)表達(dá);2)由于在同一個共表達(dá)網(wǎng)絡(luò)中的基因可能具有相似的功能,通過亞網(wǎng)絡(luò)的基因聚類,我們推測出幾個噬菌體未知基因可能的生物學(xué)功能;3)宿主的氨基酸代謝和小分子轉(zhuǎn)運功能相關(guān)基因是最容易受到噬菌體調(diào)控的基因。噬菌體PaP3基因的重新注釋:綜合利用六個基因預(yù)測平臺對噬菌體PaP3的基因進(jìn)行重新預(yù)測分析,并利用RNA-seq對預(yù)測出來的基因進(jìn)行表達(dá)驗證。最終將噬菌體PaP3的基因由原來預(yù)測的71個基因補充至126個基因。抑菌基因orf70.1及其靶蛋白的鑒定:根據(jù)前期工作,我們從兩個能與宿主蛋白相互作用的噬菌體蛋白中,鑒定出一個能夠編碼抑菌蛋白的基因—orf70.1,其編碼產(chǎn)物命名為gp70.1(geneaP3product,gp)。在gp70.1表達(dá)的情況下,宿主菌PA3在固體培養(yǎng)基上呈現(xiàn)針尖樣小菌落,在液體培養(yǎng)基中生長周期被延遲。生物信息分析和BLASTP顯示,gp70.1是一個新的未知功能的蛋白,預(yù)測小大為13kd左右,不存在保守結(jié)構(gòu)域,在已知的蛋白數(shù)據(jù)庫中沒有檢測到有意義的同源蛋白。我們純化了gp70.1,其大小與生物信息預(yù)測相符。此外我們還制備了gp70.1的多克隆抗體,利用GST-pullaP3down實驗,證實了gp70.1能在體外與RpoaP3S直接結(jié)合。Gp70.1對宿主菌RpoaP3S功能的影響:RpoaP3S是銅綠假單胞菌中重要的應(yīng)激條件全局性轉(zhuǎn)錄調(diào)控因子,分析結(jié)果顯示,RpoaP3S與gp70.1的相互作用能抑制RpoaP3S調(diào)控的應(yīng)激反應(yīng),生物膜形成以及毒力等。多種方法聯(lián)用綜合分析抑菌蛋白gp70.1對銅綠假單胞菌的影響:聯(lián)合表達(dá)譜芯片技術(shù),RT-qaP3PCR,基于核磁共振質(zhì)譜(NMR)的代謝組分析以及一系列表型分析,我們綜合考察了gp70.1對銅綠假單胞菌PA3的影響。結(jié)果顯示,在細(xì)菌生長的延遲期,對數(shù)期和穩(wěn)定期一共有178個宿主菌基因受到gp70.1的影響,這些差異表達(dá)的基因主要涉及細(xì)菌的細(xì)胞外功能和代謝功能。接著,NMR分析發(fā)現(xiàn)gp70.1對細(xì)菌的氨基酸代謝具有顯著的抑制作用。表型分析顯示,gp70.1對PA3的基本形態(tài)沒有影響,但是能強烈地抑制細(xì)菌的胞外蛋白酶,胞外多糖,胞外纖維素酶,綠膿色素以及運動功能。綜上所述,我們對銅綠假單胞菌噬菌體PaP3對宿主菌PA3全基因組轉(zhuǎn)錄的影響有了深入的了解,并繪制的基因水平的噬菌體PaP3-宿主相互作用網(wǎng)絡(luò),為后續(xù)的噬菌體抗菌蛋白的篩選以及噬菌體-宿主相互作用的深入解析提供了研究基礎(chǔ)。此外我們對噬菌體PaP3基因的補充注釋,并在此基礎(chǔ)上鑒定了一個新的噬菌體PaP3編碼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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R378
，
本文編號：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R378
，
本文編號：2172445
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