ZnuA基因缺失对致病性大肠杆菌生物学特性的影响

Effects of ZnuA gene deletion on the biological characteristics of pathogenic Escherichia coli

目的为研究Zn 2+转运系统ZnuABC关键分子ZnuA对致病性大肠杆菌生物特性的影响。方法利用λRed同源重组方法构建大肠杆菌CL-1菌株ZnuA基因缺失菌株,对比ZnuA基因缺失前后大肠杆菌生长、生化特性的改变,以及大肠杆菌的生物被膜产生、抗生素敏感性及其对结肠腺癌细胞的黏附能力的差异。结果成功构建大肠杆菌CL-1菌株ZnuA基因缺失菌株CL-1ΔZnuA和回补菌株CL-1ΔZnuA/pZnuA,生长曲线结果显示,在低浓度Zn 2+条件下,CL-1ΔZnuA的生长明显弱于野生型菌株和回补菌株,但生化特性无改变。生物被膜形成测定试验结果显示,ZnuA基因缺失能够减少40%大肠杆菌生物被膜形成能力,并明显增加大肠杆菌对氧氟沙星的敏感性。黏附试验结果显示,在细菌培养时添加螯合剂处理,缺失菌株CL-1ΔZnuA对CaCO-2细胞的黏附能力明显下降。小鼠模型显示,ZnuA基因缺失会引起大肠杆菌毒力的显著下降。结论锌获取蛋白ZnuA的缺失会导致大肠杆菌多种生物特性的改变,揭示了锌元素对致病性大肠杆菌致病的重要作用,为后续大肠杆菌病的防控提供了依据。

The roles of ZnuA,the key molecule in the Zinc uptake system,ZnuABC,on the biological characteristics of pathogenic Escherichia coli were determined.The ZnuA-deficient E.coli CL-1 mutant strain was generated by utilizing theλRed homologous recombination method.On this basis,the effects of ZnuA deletion on the growth and biochemical characteristics of E.coli were evaluated,and the biofilm forming ability,antibiotic sensitivity,and adhesion ability to the E.coli Caco-2 cell line were analyzed.The results showed that the CL-1ΔZnuA mutant with a ZnuA gene deletion and responding complement strain,CL-1ΔZnuA/pZnuA,was successfully constructed.The CL-1ΔZnuA mutant showed no difference in biochemical characteristics,but apparently retarded growth when under a low Zn 2+concentration,compared with CL-1 and CL-1ΔZnuA/pZnuA strains.The results of biofilm formation assay showed that ZnuA knockout led to an approximate 40%decrease in biofilm formation,and apparently increased the sensitivity to ofloxa cin.The CL-1ΔZnuA mutant showed impaired adhesion ability to CaCO-2 cells with pretreatment of TPEN,a chelator of Zn 2+,compared with CL-1 and CL-1ΔZnuA/pZnuA strains.The decreased virulence of CL-1ΔZnuA mutant was determined using an LD 50 assay in a mouse model.Deletion of the zinc capture protein,ZnuA,will lead to changes in various biological characteristics of E.coli,demonstrating the important role of zinc in the pathogenesis of E.coli,and providing a basis for the prevention and control of pathogenic E.coli.