自转运丝氨酸蛋白酶在猪源肠外致病性大肠杆菌致病过程中的作用  

The pathogenic roles of serine protease autotransporters in porcine originated extraintestinal pathogenic Escherichia coli

作　　者：章雅婷 泮欣铭 陈蓉 马家乐[1] 姚火春[1] ZHANG Yating;PAN Xinming;CHEN Rong;MA Jiale;YAO Huochun(College of Veterinary Medicine,OIE Reference Laboratory for Swine Streptococosis,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院/南京农业大学猪链球菌病OIE参考实验室,江苏南京210095

出　　处：《畜牧与兽医》2023年第6期93-100,共8页Animal Husbandry & Veterinary Medicine

基　　金：国家自然基金青年项目(31802187)。

摘　　要：为了探究猪源肠外致病性大肠杆菌(ExPEC)的致病机制,以自转运丝氨酸蛋白酶(SPAT)为关注点,构建了单基因和双基因缺失株,通过亚细胞定位、红细胞凝集及黏附、黏蛋白裂解和小鼠感染等试验,探究SPAT1和SPAT2在猪源ExPEC致病过程中的作用。结果显示:猪源ExPEC强毒菌株PU-1分别在染色体及毒性质粒上编码SPAT1和SPAT2;通过间接免疫荧光和Western blot检测发现2个蛋白表达于细菌表面且可分泌到培养上清液;血凝试验和红细胞黏附ELISA检测发现,SPAT1和SPAT2可引起红细胞凝集,并黏附于红细胞表面;黏蛋白裂解试验和流式细胞术分析发现,SPAT1和SPAT2可裂解宿主呼吸道黏蛋白以及中性粒细胞表面糖蛋白CD44和CD93;小鼠感染试验发现,SPAT1和SPAT2双基因缺失导致菌株毒力显著降低,且小鼠血液载菌量大幅下降,中性粒细胞数量明显上升。本研究鉴定的2个SPAT可作为防控ExPEC引起猪肠道外感染的潜在药物靶标,为防控猪群ExPEC感染方案的制定提供参考。To investigate the pathogenic mechanisms of porcine extraintestinal pathogenic Escherichia coli(ExPEC),this work focused on the serine-protease autotransporters(SPATs),and then constructed their deletion mutants in a clinical strain for the following study.The roles of SPAT1 and SPAT2 proteins in the pathogenesis of porcine ExPEC were investigated by analyzing the subcellular localization,erythrocyte agglutination and binding,mucin lysis and mouse infection models.The results showed that the porcine ExPEC virulent strain PU-1 encoded two serine-protease autotransporters,SPAT1 and SPAT2,on the chromosome and the virulent plasmid,respectively.The indirect immunofluorescence assay and Western blot analyses found that both proteins were exported to the bacterial surface or released to the extracellular supernatant,suggesting their potential roles during the bacterial infection by contacting with host components or cells.The results of the hemagglutination assay and the erythrocyte binding ELISA assay showed that both proteins could adhere to the surface of erythrocyte and cause agglutination,suggesting that these proteins promoted the bacterial spread and proliferation by adhering to erythrocyte,and mediated the immune escape.The mucin lysis assay and flow cytometry analysis showed that the host respiratory mucin and the neutrophil surface glycoproteins CD44 and CD93 were cleaved by the proteins SPAT1 and SPAT2,which uncovered their functions in mucin lysis and in resisting the clearance of host antibacterial action.The mouse infection found that the virulence of the double deletion mutant ΔSPAT1&2 was significantly decreased,as compared with the wild-type strain,and the bacterial load in blood was also significantly reduced;while the quantity of neutrophils increased conspicuously during the infection,which indicated that these two essential virulence factors might cause the immunosuppression of the host.In conclusion,the two identified SPATs might serve as therapeutic targets for porcine ExPEC,providing potential

关 键 词：肠外致病性大肠杆菌 自转运丝氨酸蛋白酶 毒力 免疫逃避 

分 类 号：S852.6[农业科学—基础兽医学]