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

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.