猪链球菌WapA毒素功能鉴定  

Functional identification of WapA toxins in Streptococcus suis

作　　者：范汝慧 白乾坤 泮欣铭 刘嘉楠 马家乐[1] 于勇[1] 姚火春[1] FAN Ruhui;BAI Qiankun;PAN Xinming;LIU Jianan;MA Jiale;YU Yong;YAO Huochun(College of Veterinary Medicine/OIE Reference Laboratory for Streptococcosis suis,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》2024年第7期85-94,共10页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(31972650)。

摘　　要：胞壁相关蛋白A(WapA)广泛存在于革兰阳性菌中,在细菌致病过程中发挥重要作用。为探究WapA的致病机制和致病作用,本研究对猪链球菌WUSS351株基因组进行分析,发现了2个编码WapA毒素的基因簇,编码的毒素分别命名为WapA1和WapA2,在GenBank数据库中检索到更多的WapA1和WapA2同源体,通过对它们的C端和N端结构域的系统进化分析,发现猪链球菌中广泛存在编码该毒素的基因簇,提示其可能具有重要的功能。后续通过构建多个WapA毒素的pBAD-His和pBAD-pleB重组质粒在大肠杆菌中表达WapA,结合生长曲线与活菌计数结果发现,WapA1-1对大肠杆菌具有周质毒性,而WapA1-2、WapA1-3、WapA2-1、WapA2-3对大肠杆菌具有胞质毒性,初步确定了WapA的毒性作用。进一步构建wapA1-1、wapA1-2、wapA2-1、wapA2-3基因缺失株(ΔwapA1-1、ΔwapA1-2、ΔwapA2-1、ΔwapA2-3),并通过竞争试验和动物感染试验发现,WapA可介导猪链球菌WUSS351株的竞争生长,并在猪链球菌致病过程中发挥重要作用。本研究结果对于猪链球菌的致病机理研究具有指导意义,为后续疾病治疗和预防提供参考。WapA is widely found in Gram-positive bacteria and plays an important role in bacterial pathogenesis.To explore the pathogenic mechanism and role of WapA,we analyzed the genome of the Streptococcus suis strain WUSS351 and identified two gene clusters encoding WapA toxins,named WapA1 and WapA2,respectively.Additional WapA1 and WapA2 homologs were retrieved in the Genbank database,and phylogenetic analysis of their C-terminal and N-terminal domains revealed a widespread cluster of genes encoding this toxin in S.suis,suggesting that it might have important functions.Subsequently,recombinant plasmids pBAD-His and pBAD-pleB containing multiple WapA toxins were constructed to express WapA in E.coli.Combined with the growth curve and viable count results,we found that WapA1-1 was periplasmic toxic to E.coli.However,WapA1-2,WapA1-3,WapA2-1 and WapA2-3 were cytosolic toxic to E.coli,and the toxic effect of WapA was preliminarily determined.The deletion strains of wapA1-1,wapA1-2,wapA2-1 and wapA2-3 genes(ΔwapA1-1,ΔwapA1-2,ΔwapA2-1 andΔwapA2-3)were further constructed.WapA could mediate the competitive growth of S.suis strain WUSS351 and play an important role in the pathogenesis of S.suis.The identification of two new toxins,WapA1 and WapA2,in S.suis in this study provided guidance for future study of the pathogenesis of S.suis,and would serve as reference for the subsequent treatment and prevention of diseases.

关 键 词：猪链球菌 WapA 毒性 致病力 

分 类 号：S855.11[农业科学—临床兽医学]