摘要
为研究丝氨酸-苏氨酸激酶(yihE)在肠炎沙门菌致病中的作用,本研究利用λ-Red同源重组技术构建了肠炎沙门菌C50336(WT)yihE基因缺失突变株C50336ΔyihE(KO)、基因回补株C50336ΔyihE/pBR322-yihE(RS),测定了WT、KO、RS菌株的生长特性、生化特性、运动能力,结果显示,与WT相比,KO株的生长速度、运动能力均无明显差异;但半乳糖酸盐利用、葡萄糖苷酶、葡萄糖醛酸酶等指标发生变化。体外模拟并检测各种应激条件下肠炎沙门菌的生长情况,结果显示,与WT相比,KO株抗多种应激能力均有不同程度下降;通过试管法、微量法等分析评估3株菌生物被膜(BF)的形成能力,另外,将各菌株分别接种至含刚果红和荧光增白剂的培养基,通过观察各菌落形态分析其BF成分的变化情况,结果显示,与WT菌株相比,KO株BF形成能力下降了约50%,但BF主要的组成成分卷毛蛋白和纤维素无明显变化。对3种菌株耐药性和KO、WT株毒力因子携带情况经RT-qPCR进行检测,结果显示,与WT相比,KO株对喹诺酮类、头孢曲松的敏感性提高;且csgA、bscA、flgG、sipB、sodc毒力基因的转录水平差异较大,而其他毒力基因的转录水平差异较小。利用KO、WT株菌株分别感染小鼠进行致病力试验,结果显示,WT株的LD_(50)为1.2×10^(5)cfu,KO株的LD_(50)为1.12×10^(6)cfu,毒力下降了约10倍。利用Over-lap PCR方法突变yihE蛋白217位天冬氨酸,利用蛋白质丝氨酸/苏氨酸激酶检测试剂盒检测其激酶活性,结果显示yihE蛋白突变后其激酶活性下降了约30%,表明217位天冬氨酸对yihE蛋白的活性非常关键。本研究证实yihE参与了肠炎沙门菌的多个生物学过程,且yihE基因的缺失能够使肠炎沙门菌的毒力下降,该结果为进一步研究沙门菌致病机制奠定了基础。
To study the role of yihE,a serine threonine kinase,in the pathogenicity of Salmonella enteritis,the yihE gene deletion mutant C50336ΔyihE was constructed byλ-Red homologous reengineering and conresponding complement strain C50336ΔyihE/PBR322-yihE(RS)was also generated for comparison of growth,motility and biochemical characteristics.The results showed that yihE-deficient mutant displayed no significant difference in the growth rate and motility compared with the wildtype C50336 strain,while it lost the ability to utilize galactolactate and express glucosidase and glucuronase in Salmonella enteritis.Knockout of yihE led to apparently decreased survival of variable extent under various stress conditions in models in vitro.Impairment of biofilm formation was also observed in yihE-deletion mutant but without significant change on expression of curl protein and cellulose,the main component of bacterial biofilm.Pathogenicity test with mice model was performed to access yihEknockout caused effect,the data showed that the LD_(50)of WT and KO strain was 1.2×10^(5)cfu and 1.12×10^(6)cfu respectively,demonstrating a 10-fold decrease in virulence.The differences in the expression of csgA,bscA,flgG,sipB and sodc genes between WT and KO strains were determined by RT-qPCR analysis.The increased sensitivity to ceftriaxone and quinolones was found after yihE deletion in Salmonella.The kinase activity assay showed that the kinase activity of yihE protein decreased by about 30%with amino acid mutation at 217 L-Aspartic acid,indicating its critical role.This study confirmed that yihE gene is involved in multiple biological processes of Salmonella enteritidis,and the deletion of yihE gene can reduce the virulence of Salmonella enteritidis,which laid a foundation for further research on the pathogenic mechanism of Salmonella enteritidis.
作者
刘畅
张雪佳
王利丽
白和平
王苗
李浩
刘勃兴
史秋梅
吴同垒
张志强
LIU Chang;ZHANG Xue-jia;WANG Li-li;BAI He-ping;WANG Miao;LI Hao;LIU Bo-xing;SHI Qiu-mei;WU Tong-lei;ZHANG Zhi-qiang(Hebei Normal University of Science&Technology,Qinhuangdao 066004,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第4期369-376,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(31802186)
河北省自然科学基金项目(C2019407070)
河北省高等学校科学技术研究项目(BJ2021055)
河北省重点研发计划项目(19226629D)。
关键词
肠炎沙门菌
丝氨酸-苏氨酸激酶
致病力
抗应激能力
生物被膜
Salmonella enteritidis
serine threonine kinase
virulence
the ability to resist stress
biological characteristics
