深度测序技术对羊布鲁菌16M株感染小鼠巨噬细胞转录组学的研究
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摘要
布鲁菌病（简称布病）是由布鲁菌（Brucella）引起的一种人兽共患性传染病，该病迄今已有一百多年的历史。布鲁菌缺乏经典的致病因子，例如外毒素、荚膜、菌毛、鞭毛、溶细胞素、胞外酶、外分泌蛋白酶等。但布鲁菌作为胞内寄生菌，可以在宿主细胞、特别是巨噬细胞中长期生存，并且可以逃逸宿主细胞的免疫防御机制，说明布鲁菌有自己独特的致病机制。
本试验从感染宿主角度展开研究，首次利用深度测序技术对布鲁菌感染小鼠巨噬细胞RAW264.7的转录组学轮廓进行了分析。通过布鲁菌胞内感染试验、细胞毒性试验、间接免疫荧光试验最终确立MOI为1000时为最佳感染条件。提取感染细胞样本总RNA经深度测序技术发现：在感染后4h，3576个差异表达基因中的58%表现上调；在感染后24h，3962个差异表达基因中的45%表现上调；并对差异表达基因进行了荧光定量PCR验证。通过GO Term、KEGG分析，在感染后24h内显著富集的通路包括溶酶体信号通路、Fc γR-介导的吞噬通路、与内质网蛋白加工相关通路，在布鲁菌的胞内吞噬、定殖和成熟方面发挥重要作用；凋亡通路、p53信号通路与细胞凋亡有关；NOD受体、B细胞受体、toll样受体(TLR)信号通路都是与免疫相关的。
试验中发现，布鲁菌感染小鼠巨噬细胞之后，引起溶酶体代谢途径中Acp5(acid phosphatase5)基因显著性变化，Acp5作为运送Man-6-P的候选酶之一，在转运溶酶体蛋白Man-6-P特异标识方面发挥重要作用。溶酶体内的水解酶要发挥作用，必须对溶酶体标识蛋白Man-6-P进行翻译后的修饰。Acp5的过表达可以导致溶酶体蛋白Man-6-P的去磷酸化效率显著增加；Acp5的下调表达可以导致Man-6-P糖蛋白的表达水平升高。我们通过RNAi技术，抑制该基因在宿主细胞的表达，评价对布鲁菌致病性的影响，发现布鲁菌的胞内增殖能力受到抑制。
本研究建立了布鲁菌感染小鼠巨噬细胞后的差异表达基因数据库，对小鼠巨噬细胞感染布鲁菌后的动态转录组学轮廓进行了描述。宿主细胞对于病原菌的调节，不是一个基因在发挥作用，而是一个有机调控的网络在发挥作用。这些差异表达基因的数据为进一步研究宿主在布鲁菌感染过程中发挥的作用提供了基础。
Brucellosis,which is caused by Brucella, is a worldwide zoonotic infectiousdisease and it has been one hundred years. Brucella lack classic pathogenic factors,such as toxins, capsule, pilus, flagellum, cytolysin, extracellular enzyme and so on.But Brucella, which is intracellular, can survival in host cell and escape the defensemechanism of host. It indicated that Brucella has the special pathogenic mechanism.
In current study, the transcriptional profile of RAW264.7cells infected with theBrucella melitensis strain16M was investigated using a technology based on deepsequencing for the first time. A suitable infection conditions was establishedaccording to the intracellular infection assay, macrophage cytotoxicity assay andindirect immunofluorescent assay when MOI is1000. Extracting RNA of infectioncell samples we found that there are approximately58%genes expressedup-regulation in which3576significantly changed genes4h post infection andapproximately45%genes expressed up-regulation in which3962significantlychanged genes24h post infection with the technology based on deep sequencing. Andwe verified the genes expressed significance difference with the real time RT-PCR.The passways significantly enriched postinfection include lysosome pathway, Fcgamma R-mediated phagocytosis and protein processing in the endoplasmicreticulum,which play an important role in phagocytosis, colonization and maturity.Apotosis pathway and p53signaling pathway which are related to apotosis. NOD-likereceptor signaling pathway, B-cell receptor and toll-like receptor signaling pathwayswhich are related to immunity.
In the study, Acp5of lysosome was changed significally postinfection. Acp5whichis one of candicate enzyme transporting Man-6-P plays an important role intransporting specific mark of lysosome protein Man-6-P. If the hydrolysis enzyme inthelysosome want to work, the marker protein of Man-6-P must be posttranslationalmodification. Overexpression of Acp5can cause the phosphorylation efficiency oflysosome protein significantly increase and expression of Man-6-P glycoprotein ismore increased if the expressiμμμon of Acp5is inhibited. So we inhibited theexpression of Acp5utilized the technology of RNAi to evaluation the effect with the infection of Brucella.
In current study, we established a database, which is based on significant differentexpression genes of mouse macrophage infected Brucella, which would be providethe basis for evaluating the role of host during the infection.
本试验从感染宿主角度展开研究，首次利用深度测序技术对布鲁菌感染小鼠巨噬细胞RAW264.7的转录组学轮廓进行了分析。通过布鲁菌胞内感染试验、细胞毒性试验、间接免疫荧光试验最终确立MOI为1000时为最佳感染条件。提取感染细胞样本总RNA经深度测序技术发现：在感染后4h，3576个差异表达基因中的58%表现上调；在感染后24h，3962个差异表达基因中的45%表现上调；并对差异表达基因进行了荧光定量PCR验证。通过GO Term、KEGG分析，在感染后24h内显著富集的通路包括溶酶体信号通路、Fc γR-介导的吞噬通路、与内质网蛋白加工相关通路，在布鲁菌的胞内吞噬、定殖和成熟方面发挥重要作用；凋亡通路、p53信号通路与细胞凋亡有关；NOD受体、B细胞受体、toll样受体(TLR)信号通路都是与免疫相关的。
试验中发现，布鲁菌感染小鼠巨噬细胞之后，引起溶酶体代谢途径中Acp5(acid phosphatase5)基因显著性变化，Acp5作为运送Man-6-P的候选酶之一，在转运溶酶体蛋白Man-6-P特异标识方面发挥重要作用。溶酶体内的水解酶要发挥作用，必须对溶酶体标识蛋白Man-6-P进行翻译后的修饰。Acp5的过表达可以导致溶酶体蛋白Man-6-P的去磷酸化效率显著增加；Acp5的下调表达可以导致Man-6-P糖蛋白的表达水平升高。我们通过RNAi技术，抑制该基因在宿主细胞的表达，评价对布鲁菌致病性的影响，发现布鲁菌的胞内增殖能力受到抑制。
本研究建立了布鲁菌感染小鼠巨噬细胞后的差异表达基因数据库，对小鼠巨噬细胞感染布鲁菌后的动态转录组学轮廓进行了描述。宿主细胞对于病原菌的调节，不是一个基因在发挥作用，而是一个有机调控的网络在发挥作用。这些差异表达基因的数据为进一步研究宿主在布鲁菌感染过程中发挥的作用提供了基础。
Brucellosis,which is caused by Brucella, is a worldwide zoonotic infectiousdisease and it has been one hundred years. Brucella lack classic pathogenic factors,such as toxins, capsule, pilus, flagellum, cytolysin, extracellular enzyme and so on.But Brucella, which is intracellular, can survival in host cell and escape the defensemechanism of host. It indicated that Brucella has the special pathogenic mechanism.
In current study, the transcriptional profile of RAW264.7cells infected with theBrucella melitensis strain16M was investigated using a technology based on deepsequencing for the first time. A suitable infection conditions was establishedaccording to the intracellular infection assay, macrophage cytotoxicity assay andindirect immunofluorescent assay when MOI is1000. Extracting RNA of infectioncell samples we found that there are approximately58%genes expressedup-regulation in which3576significantly changed genes4h post infection andapproximately45%genes expressed up-regulation in which3962significantlychanged genes24h post infection with the technology based on deep sequencing. Andwe verified the genes expressed significance difference with the real time RT-PCR.The passways significantly enriched postinfection include lysosome pathway, Fcgamma R-mediated phagocytosis and protein processing in the endoplasmicreticulum,which play an important role in phagocytosis, colonization and maturity.Apotosis pathway and p53signaling pathway which are related to apotosis. NOD-likereceptor signaling pathway, B-cell receptor and toll-like receptor signaling pathwayswhich are related to immunity.
In the study, Acp5of lysosome was changed significally postinfection. Acp5whichis one of candicate enzyme transporting Man-6-P plays an important role intransporting specific mark of lysosome protein Man-6-P. If the hydrolysis enzyme inthelysosome want to work, the marker protein of Man-6-P must be posttranslationalmodification. Overexpression of Acp5can cause the phosphorylation efficiency oflysosome protein significantly increase and expression of Man-6-P glycoprotein ismore increased if the expressiμμμon of Acp5is inhibited. So we inhibited theexpression of Acp5utilized the technology of RNAi to evaluation the effect with the infection of Brucella.
In current study, we established a database, which is based on significant differentexpression genes of mouse macrophage infected Brucella, which would be providethe basis for evaluating the role of host during the infection.
引文
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