鼠疫耶尔森菌201菌株中重要代谢相关基因特定突变与其小鼠毒力的关系研究

Specific mutations in certain metabolism-related genes of Yersinia pestis strain 201 and their effects on the virulence to mice

目的探讨布氏田鼠型鼠疫耶尔森菌(简称鼠疫菌) 201株asp A和gln K的单核苷酸多态性位点(SNP)及fyu A三碱基缺失与小鼠毒力的关系。方法通过基于自杀载体p DS132同源重组的无痕突变技术,分别构建了鼠疫菌201株的asp A和gln K基因的SNP位点突变株(201-asp ATTG和201-gln KCCA)以及fyu A的GCA突变株(201-fyu AGCAdel)。随后采用腹股沟皮下注射方式对小鼠攻毒,通过生存曲线确定以上基因中的突变对小鼠毒力是否具有显著影响。结果生存曲线结果显示,201-asp ATTG和201-gln KCCA两株突变菌与野生型对小鼠毒力无明显差异; 201-fyu AGCAdel突变株在2周内不能对小鼠致死,与野生型毒力存在显著差异,且其质粒回补株对小鼠毒力恢复到野生型水平。结论该研究中所涉及的asp A和gln K的SNP突变不能使鼠疫菌201株对小鼠明显减毒,asp A和gln K的特定位点突变导致的蛋白活性差异可能与鼠疫菌201株对小鼠毒力无关,而fyu A的GCA缺失可导致鼠疫菌对小鼠的毒力明显下降,有必要对其内在机制进行深入研究。

Objective To obtain mutants of Yersinia pestis strain 201 harboring specific single nucleotide polymorphisms(SNPs)in asp A and gln K,and a GCA-deletion in fyu A,and then to explore whether these mutations affect the virulence to mice.Methods By using scarless genetic modification technique based on suicide plasmid p DS132,three mutants harboring target mutations were constructed:SNP within asp A(201-asp ATTG)and gln K(201-gln KCCA),and the GCAdeletion within fyu A(201-fyu AGCAdel).A complementary strain of the fyu A mutant was also constructed(201-fyu A-Comp).Then,BALB/c mice were challenged by the mutants and the complementary strain via subcutaneous injection.The survival curves were used to determine the effects of the target mutations on the virulence to mice.Results Survival curves revealed that 201-asp ATTGand 201-gln KCCAkilled all the mice within two weeks,so there was no significant difference between these two mutants and the wild type.However,no challenged mouse was killed by 201-fyu AGCAdelin two weeks,indicating a significant difference between the fyu A-GCA-deletion mutant and the wild type.Meanwhile,the complementary strain restored the virulence to mice,killing all the mice.Conclusion The tested SNP of asp A or gln K in Y.pestis seems to have no effect on its virulence to mice,indicating that the activity of Asp A or Gln K might be unrelated to the pathogenesis process of Y.pestis strain 201 in mice.However,the deletion of GCA within fyu A could lead to a significant attenuation on virulence of Y.pestis in mice,which deserves further investigation.