The effects of NDM-5 on Escherichia coli and the screening of interacting proteins

Abstract

Carbapenem-resistant Escherichia coli ( E. coli ) strains are widely distributed and spreading rapidly, creating significant challenges for clinical therapeutics. NDM-5, a novel mutant of New Delhi Metallo-β-Lactamase-1 (NDM-1), exhibits high hydrolase activity toward carbapenems. Since the genetic backgrounds of clinically isolated carbapenem-resistant E. coli are heterogeneous, it is difficult to accurately evaluate the impact of bla NDM–5 on antibiotic resistance. Herein, E. coli BL21 was transformed with a plasmid harboring bla NDM–5 , and the resultant strain was named BL21 (pET-28a- bla NDM–5 ). Consistent with the findings of previous studies, the introduction of exogenous bla NDM–5 resulted in markedly greater resistance of E. coli to multiple β-lactam antibiotics. Compared with BL21 (pET-28a), BL21 (pET-28a- bla NDM–5 ) exhibited reduced motility but a significant increase in biofilm formation capacity. Furthermore, transcriptome sequencing was conducted to compare the transcriptional differences between BL21 (pET-28a) and BL21 (pET-28a- bla NDM–5 ). A total of 461 differentially expressed genes were identified, including those related to antibiotic resistance, such as genes associated with the active efflux system ( yddA , mcbR and emrY ), pili ( csgC , csgF and fimD ), biofilm formation ( csgD , csgB and ecpR ) and antioxidant processes ( nuoG ). Finally, the pGS21a plasmid harboring bla NDM–5 was transformed into E. coli Rosetta2, after which the expression of the NDM-5 protein was induced using isopropyl-β-D-thiogalactoside (IPTG). Using glutathione-S-transferase (GST) pull-down assays, total proteins from E. coli were scanned to screen out 82 proteins that potentially interacted with NDM-5. Our findings provide new insight into the identified proteins to identify potential antibiotic targets and design novel inhibitors of carbapenem-resistant bacteria.