幽门螺杆菌东亚型菌株GZ7/cagA^(+)和GZ7/ΔcagA源外膜囊泡的蛋白组学比较  

Proteomic comparison of outer membrane vesicles of Helicobacter pylori GZ7/cagA^(+)and GZ7/△cagA

作　　者：彭国玲 周佳 廖永慧 谢渊 周建奖[1,2,3] 赵艳 PENG Guoling;ZHOU Jia;LIAO Yonghui;XIE Yuan;ZHOU Jianjiang;ZHAO Yan(School of Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China;Key Laboratory of Molecular Biology,Guizhou Medical University,Guiyang 550004,Guizhou,China;Key Laboratory of Endemic and Ethnic Diseases,Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院,贵州贵阳550025 [2]贵州医科大学分子生物学重点实验室,贵州贵阳550004 [3]贵州医科大学地方病与少数民族疾病教育部重点实验室,贵州贵阳550004

出　　处：《贵州医科大学学报》2024年第5期636-644,共9页Journal of Guizhou Medical University

基　　金：国家自然科学基金(32160166);国家自然科学基金(82260405);国家自然科学基因培育项目(20NSP068)。

摘　　要：目的通过分离、鉴定和比较细胞毒素相关基因A蛋白(cagA)、阳性幽门螺杆菌(H.pylori)、东亚型菌株GZ7/cagA^(+)及其cagA敲除菌株GZ7/ΔcagA来源的外膜囊泡(OMVs)中的差异表达蛋白(DEPs),分析cagA基因对OMVs中蛋白表达的影响。方法采用超速离心法分别提取GZ7/ΔcagA和GZ7/cagA^(+)的OMVs,通过透射电镜和纳米颗粒追踪技术鉴定其形态和粒径,使用Western blot技术验证两组OMVs中cagA蛋白的表达,分析OMVs的蛋白质组学;对蛋白组学数据进行质控分析和主成分分析鉴定后,以上调蛋白倍数变化(FC)>2.0、下调蛋白FC<0.5,FDR≤0.05为筛选条件筛选DEPs,利用OmicsBean在线工具、Gene Ontology和KOBAS对DEPs进行生物信息学分析;采用免疫荧光鉴定OMVs细胞在细胞中的定位,实时无标记细胞分析仪检测细胞活性。结果通过电镜和粒径证实成功分离纯化了OMVs;蛋白质组分析发现,GZ7/cagA^(+)-OMVs组与GZ7/ΔcagA-OMVs组比较有79个DEPs,其中38个蛋白下调、41个蛋白上调;生物信息学分析显示,DEPs主要与丙酮酸代谢、丙酸代谢、糖酵解/糖异生及柠檬酸循环等代谢途径有关;免疫荧光和实时无标记细胞分析证实H.pylori来源的OMVs能进入细胞并定位在线粒体并抑制细胞增殖。结论cagA能影响H.pylori分泌的OMVs中蛋白质的成分,DEPs可能促进cagA^(+)H.pylori在胃黏膜上的定植及致病性。Objective To investigate the effects of cyto toxin associated gene A protein(cagA)gene on protein expression in outer membrane vesicles(OMVs)by isolating,identifying and comparing differentially expressed proteins(DEPs)in cagA,positive Helicobacter pylori(H.pylori),East Asian strain GZ7/cagA^(+)and cagA knockout strain GZ7/ΔcagA-derived OMVs.Methods The OMVs of GZ7/ΔcagA and GZ7/cagA^(+)were extracted by ultracentrifugation,and their morphology and particle size were identified by transmission electron microscopy and nanoparticle tracking technology.The expression of cagA protein in the two groups of OMVs was verified by Western blot,and the proteomics of OMVs was analyzed.After quality control analysis and principal component analysis of proteomic data,DEPs were screened under the screening conditions of up-regulated protein fold change(FC)>2.0,down-regulated protein FC<0.5,FDR≤0.05,and bioinformatics analysis of DEPs was performed using OmicsBean online tool,Gene Ontology and KOBAS.The localization of OMVs cells in cells was identified by immunofluorescence,and the cell activity was detected by real-time cell analyzer(RTCA).Results The successful separation and purification of OMVs were confirmed by electron microscopy and particle size.Proteome analysis showed that there were a total of 79 DEPs in the GZ7/cagA^(+)-OMVs group compared with the GZ7/ΔcagA-OMVs group,of which 38 proteins were down-regulated and 41 proteins were up-regulated.Bioinformatics analysis showed that DEPs were mainly related to metabolic pathways such as pyruvate metabolism,propionic acid metabolism,glycolysis/gluconeogenesis and citric acid cycle.Immunofluorescence and RTCA confirmed that H.pylori-derived OMVs could enter cells and locate in mitochondria and inhibit cell proliferation.Conclusion The present study demonstrates that cagA can affect the protein composition of OMVs secreted by H.Pylori and DEPs may promote the colonization and pathogenicity of cagA^(+)H.pylori on gastric mucosa.

关 键 词：幽门螺杆菌 细胞毒素相关基因A蛋白 胃癌 蛋白组 差异表达蛋白 线粒体 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]