禽致病性大肠杆菌外膜囊泡（OMVs）关键效应蛋白的鉴定及其致巨噬细胞损伤的机制研究

Avian Pathogenic Escherichia coli (APEC) is an important pathogen of poultry. These APEC-associated systemic infections have been proven economically devastating to global poultry industries, and APEC might hold zoonotic potential. Intracellular macrophage infection is a key part of APEC pathogenicity during the early stages of avian colibacillosis. Research currently available is not sufficient to reveal the molecular mechanisms for APEC-induced macrophage injury. Identifying the protein expression profiling of APEC outer membrane vesicles (OMVs) acts as the research entry point in this study. Mass spectrometry-based proteomic analysis is used to reveal the secretory protein profiling of APEC OMVs. Then, the comparative analysis of protein profile differences between high virulent and non-virulent APEC strains is performed to screen the specific proteins secreted from OMVs of APEC virulent strains. Transcriptional expression profiling of the macrophages after interaction with OMVs (isolated from high virulent or non-virulent APEC strains) will be compared by transcriptome analysis, and the major signaling pathways responsible for macrophage damage caused by OMVs of the APEC virulent strains are identified. The characteristic damage phenotypes will be revealed by a series of corresponding tests. Then, the critical effectors of AEPC OMVs that cause macrophage immune response damage are revealed by a series of molecular biological methods. And the molecular interaction mechanisms that OMVs effectors control or inhibit the macrophage immune regulatory signal will be elucidated. A better understanding of the mechanisms by which OMVs manipulates macrophage damage will contribute to anti-bacterial drugs/vaccines design to defense APEC infections.

禽致病性大肠杆菌(APEC)是禽的重要细菌性病原，严重危害养禽业，甚至具有人畜共患潜力。APEC感染巨噬细胞是其致病的一个关键环节，目前研究成果不足以阐明APEC致巨噬细胞损伤的分子机制，在已有研究的基础上,申请者拟以鉴定APEC外膜囊泡(OMVs)蛋白质表达谱为研究切入点，通过高通量质谱方法比较APEC强、弱毒株OMVs蛋白质表达谱差异，筛选APEC强毒株OMVs特有的外泌性蛋白质；在APEC强、弱毒株OMVs与巨噬细胞互作后，转录组比较分析巨噬细胞转录谱差异，探究APEC强毒株OMVs造成巨噬细胞损伤的主要信号途径，进而揭示APEC OMVs引起巨噬细胞损伤的表现形式；通过一系列分子生物学方法鉴定致巨噬细胞免疫应答损伤的OMVs关键效应蛋白，阐明OMVs关键效应蛋白操纵或抑制巨噬细胞免疫应答的分子互作机制，从而为禽致病性大肠杆菌新药物靶标的筛选及新型疫苗的开发提供一种新的思路。

肠道外致病性大肠杆菌(ExPEC)是一种能够引起人和动物疾病的重要病原菌ExPEC可以引起肠道外的多种感染，包括肺部感染、尿路感染、腹腔感染、新生儿脑膜炎和血流感染等。禽致病性大肠杆菌(APEC)是ExPEC的一种致病亚型，是感染禽类的重要细菌性病原。APEC/ExPEC给养殖业带来较大的危害，且威胁人类健康。APEC/ExPEC具有多种毒力因子，胞外囊泡(Extracellular vesicles, EVs)被认为是其重要的毒力因子，EVs通常也被称为膜囊泡(Membrane vesicles, MVs)。EVs包含外膜囊泡(Outer membrane vesicles，OMVs)、外内膜囊泡（OIMVs）和爆炸性外膜囊泡（EOMVs），其中OMVs是典型的细菌胞外囊泡(EVs)。APEC/ExPEC分泌的EVs包含蛋白质和核酸等多种物质成分。细菌可以利用这些物质逃避或破坏宿主的防御机制，促进细菌在肠道外环境中的存活和定植。本研究通过蛋白质组学分析发现ExPEC EVs具有较多的胞质蛋白和核糖体蛋白，通过电镜观察发现APEC/ExPEC EVs包含OMVs和携胞质的胞外囊泡（OIMVs和EOMVs）。通过透射电镜、WB分析和免疫荧光标记等试验发现内溶素触发的细胞膜裂解是APEC/ExPEC产生EVs的重要原因；SOS应激反应可以导致APEC/ExPEC含胞质成分EVs的总量发生动态变化。通过细胞毒性检测、线粒体膜电位检测和炎性反应相关蛋白水平检测等试验发现APEC/ExPEC EVs对巨噬细胞具有细胞毒性。携胞质EVs比例越高，APEC/ExPEC EVs导致的细胞毒性越强，并会引起更严重的线粒体损伤和细胞内源性凋亡。通过研究，我们揭示了ExPEC EVs蛋白质的主要组成成分（胞质蛋白和核糖体蛋白）；发现了内溶素介导APEC/ExPEC携胞质EVs形成的新机制。

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