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

Lin Li; Yiming Gao; Longbo Wang; Fang Lu; Qianyu Ji; Yanfang Zhang; Shuo Yang; Ping Cheng; Feifei Sun; Shaoqi Qu

doi:10.3389/fmicb.2024.1328572

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

Front Microbiol. 2024 Jan 30:15:1328572. doi: 10.3389/fmicb.2024.1328572. eCollection 2024.

Authors

Lin Li^#^{1

2}, Yiming Gao^#¹, Longbo Wang¹, Fang Lu¹, Qianyu Ji¹, Yanfang Zhang¹, Shuo Yang¹, Ping Cheng^{1

2}, Feifei Sun^{1

2}, Shaoqi Qu^{1

2}

Affiliations

¹ Pharmacology and Toxicology Laboratory, Animal-Derived Food Safety Innovation Team, College of Animal Science and Technology, Anhui Agricultural University, Hefei, China.
² Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, Anhui Agricultural University, Hefei, China.

^# Contributed equally.

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.

Keywords: Escherichia coli; GST pull-down; NDM-5; mass spectrometry; transcriptomics.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Natural Science Foundation of Anhui Province (2208085MC79); the National Natural Science Foundation of China (32302923); the Key Project of the Department of Education of Anhui Province (No. KJ2021A0147), and the Animal-Derived Food Safety Innovation Team (ANRC2021040).