新型甲氧苄啶耐药二氢叶酸还原酶DfrB7的生化分析与宿主菌耐药机制研究

Characterization and biochemistry analysis of a novel trimethoprim resistant DfrB7 and the mechanism of trimethoprim resistance

摘要:

[目的] 分析新型甲氧苄啶获得性耐药蛋白DfrB7的生化性质，探究其与B家族代表性的二氢叶酸还原酶（DHFR）对甲氧苄啶获得性耐药的生化基础。[方法] 构建系统进化树分析B家族DHFRs与新型DfrB7的进化关系。将dfr基因分别构建到pACYC184和pET15b（+）载体并转化到相应大肠杆菌中。使用微量肉汤稀释法确定克隆菌株对甲氧苄啶的耐药性。测定DHFRs使用NADPH作为质子供体催化二氢叶酸还原的酶活反应参数。通过等温滴定量热法测定甲氧苄啶的解离常数。[结果] 克隆到大肠杆菌中的新型dfrB7基因表现出对甲氧苄啶的高耐药表型。系统进化树确定了dfrB7编码B家族的DHFRs。检测并比较DfrB7和代表性DHFRs的酶活性质、抑制剂的亲和力，与染色体上DHFR相比，DfrB7与DfrB1均表现出显著降低的催化活性，通过生化实验证实B家族二氢叶酸还原酶对甲氧苄啶结合力极差。[结论] 新型dfrB7基因编码的DfrB7具有B家族二氢叶酸还原酶的普遍特征。B家族二氢叶酸还原酶赋予宿主菌对甲氧苄啶的获得性耐药与该酶对甲氧苄啶的低亲和力有关。

Abstract:

[Objective] To characterize novel acquired trimethoprim resistance gene dfrB7, and to determine the biochemical basis of acquired trimethoprim resistance for DfrB7 coded by novel dfrB7 and previously known representative Family B DHFRs.[Methods] Phylogenetic analysis of previously reported DfrB proteins and the novel DfrB7 was performed. PCR-amplified dfr genes were cloned into pACYC184 and pET15b(+) vectors, followed by transformation into Escherichia coli. The dfr-pACYC184 plasmid containing E. coli strains were tested for trimethoprim susceptibility by microdilution broth method. Enzymatic catalysis parameters were determined by analyzing the NADPH:dihydrofolate oxidoreductase activities. Isothermal titration calorimetry was performed to measure the dissociation constants between TMP and DHFRs. [Results] Novel dfrB7 gene conferred trimethoprim resistance (MIC ≥ 1024 mg/L) when it was cloned into E. coli. Phylogenetic analysis showed that dfrB7 encodes a Family B DHFR. Novel dfrB7 and previously known representative dfr genes were overexpressed and purified for the analysis of enzymatic parameters and TMP affinity. Comparing with chromosomal DHFR, both DfrB1 and DfrB7 showed significantly lower activities, and Family B DHFRs have drastic lower affinities to trimethoprim.[Conclusion] DfrB7 encoded by a novel dfrB7 gene has common characteristics of Family B DHFRs. The acquired resistance to trimethoprim is caused by the low affinities of Family B DHFRs to trimethoprim.