类鼻疽伯克霍尔德菌Ⅵ型分泌系统的Hcp1蛋白转位介导多核巨细胞形成  

Translocation of Hcp1 protein in typeⅥsecretion system of Burkholderia pseudomallei mediates formation of multinucleated giant cells

作　　者：吴潘 饶承龙 南栋琪 陈建高 张子元 刘文正 王民洋 闫晶敏 李倩[1,2] 毛旭虎[1,2] WU Pan;RAO Chenglong;NAN Dongqi;CHEN Jiangao;ZHANG Ziyuan;LIU Wenzheng;WANG Minyang;YAN Jingmin;LI Qian;MAO Xuhu(Department of Clinical Microbiology and Immunology,Faculty of Pharmacy and Medical Laboratory,Army Medical University(Third Military Medical University),Chongqing,400038,China;State Key Laboratory of Trauma and Chemical Poisoning,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]陆军军医大学(第三军医大学)药学与检验医学系临床微生物与免疫学教研室,重庆400038 [2]陆军军医大学(第三军医大学)创伤与化学中毒全国重点实验室,重庆400038

出　　处：《陆军军医大学学报》2024年第15期1721-1728,共8页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(82272350)。

摘　　要：目的探讨类鼻疽伯克霍尔德菌Ⅵ型分泌系统溶血素共调节蛋白1(hemolysin coregulatory protein 1,Hcp1)在感染宿主细胞引起多核巨细胞(multinucleated giant cells,MNGC)形成的作用及机制。方法分别采用同源重组和质粒回补技术构建类鼻疽菌hcp1基因缺失株(BPC006Δhcp1)和缺失回补株(BPC006Δhcp1::hcp1);分别用类鼻疽菌BPC006野生株、BPC006Δhcp1和BPC006Δhcp1::hcp1感染RAW264.7细胞,通过激光共聚焦分析Hcp1蛋白的定位情况;在293T细胞中转染Hcp1真核表达载体后进行细胞质膜分离进一步验证定位情况;在细胞感染模型上,通过吉姆萨染色检测抗Hcp1多克隆抗体封闭对MNGC形成的影响,探究Hcp1在类鼻疽菌感染导致的MNGC形成中的作用和生物学意义。结果Western blot检测结果显示BPC006Δhcp1不表达Hcp1蛋白,而BPC006Δhcp1::hcp1能恢复Hcp1蛋白的表达,成功构建类鼻疽菌BPC006Δhcp1和BPC006Δhcp1::hcp1。细胞免疫荧光共定位实验和质膜分离实验都表明Hcp1可以定位在宿主细胞膜上,且相较于对照组抗Hcp1多克隆抗体能抑制类鼻疽菌感染所诱导的MNGC形成(P<0.01)。结论类鼻疽菌感染RAW264.7细胞时Hcp1可转位至细胞膜,并在MNGC形成中发挥重要作用。Objective To analyze the mechanism that Hcp1 protein in typeⅥsecretion system of Burkholderia pseudomallei(B.pseudomallei)mediates the formation of multinucleated giant cells(MNGCs)when host cells are infected by the bacterium.Methods The mutant strain(BPC006Δhcp1)and complementation strain(BPC006Δhcp1::hcp1)were constructed by homologous recombination and plasmid complement technology,respectively.After RAW264.7 cells were infected with B.pseudomallei,the localization of Hcp1 in host cells was analyzed by immunofluorescence staining.The localization was further verified by cytoplasmic-membrane isolation in 293T cells after transfecting pCDNA4.1-Hcp1.The biological significance and effect of Hcp1 were explored by the anti-Hcp1 polyclonal antibody blocking and the formation of MNGC was detected by Giemsa staining.Results Western blotting showed that BPC006Δhcp1 could not express Hcp1,while BPC006Δhcp1::hcp1 restored Hcp1 expression.The above results proved that the mutant and complement strains were successfully constructed.Both cellular immunofluorescence co-localization and cytoplasmic-membrane isolation experiments showed that Hcp1 localized to host cell membranes.Last but not least,compared with the control group,anti-Hcp1 polyclonal antibodies inhibited the formation of MNGC(P<0.01).Conclusion Hcp1 protein in typeⅥsecretion system of B.pseudomallei is able to translocate to the RAW264.7 cell membranes and plays an important role in the formation of MNGCs.

关 键 词：类鼻疽伯克霍尔德菌 Hcp1 多核巨细胞 基因缺失与回补 

分 类 号：R378.99[医药卫生—病原生物学] R392.11[医药卫生—基础医学] R392.3