Highly Conserved Genetic Factors Regulating bla NDM Gene Expression
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The New Delhi metallo-β-lactamase (NDM; EC 3.5.2.6) poses significant challenges to carbapenem treatment, yet the regulatory mechanisms governing blaNDM gene expression remain poorly understood. In this study, we identified a highly conserved 110 bp sequence located upstream of the blaNDM gene by comparative analysis of 109 clinical isolates and 2,706 nucleotide sequences from the NCBI database. This conserved sequence was characterized in all available NDM variants spanning 80 bacterial species. Bioinformatics analysis revealed a core promoter (PNDM) and two transcription factor binding sites (ArcA and ArgR2) within the sequence. Mutations of the PNDM promoter significantly reduced blaNDM expression (mRNA and protein) by more than 90% (P < 0.01) and completely restored carbapenem susceptibility. Electrophoretic mobility shift assays (EMSA) confirmed the specific binding of ArcA and ArgR2 proteins to their predicted sites. Mutations in the ArcA and ArgR2 binding sites decreased blaNDM protein production with less pronounced effects compared to PNDM promoter mutations, resulting in 24% and 32.7% reductions, respectively. The findings highlight the critical role of the highly conserved 110 bp sequence in regulating blaNDM expression, offering potential targets for novel therapeutic strategies.
Abstract
The New Delhi metallo-β-lactamase (NDM; EC 3.5.2.6) poses significant challenges to carbapenem treatment, yet the regulatory mechanisms governing blaNDM gene expression remain poorly understood. In this study, we identified a highly conserved 110 bp sequence located upstream of the blaNDM gene by comparative analysis of 109 clinical isolates and 2,706 nucleotide sequences from the NCBI database. This conserved sequence was characterized in all available NDM variants spanning 80 bacterial species. Bioinformatics analysis revealed a core promoter (PNDM) and two transcription factor binding sites (ArcA and ArgR2) within the sequence. Mutations of the PNDM promoter significantly reduced blaNDM expression (mRNA and protein) by more than 90% (P < 0.01) and completely restored carbapenem susceptibility. Electrophoretic mobility shift assays (EMSA) confirmed the specific binding of ArcA and ArgR2 proteins to their predicted sites. Mutations in the ArcA and ArgR2 binding sites decreased blaNDM protein production with less pronounced effects compared to PNDM promoter mutations, resulting in 24% and 32.7% reductions, respectively. The findings highlight the critical role of the highly conserved 110 bp sequence in regulating blaNDM expression, offering potential targets for novel therapeutic strategies.