Bacterial resistance1

Description

Carbapenem-resistant Enterobacteriaceae (CRE), particularly strains producing New Delhi metallo-β-lactamase-1 (NDM-1), pose a severe global health threat. The SecYEGtranslocon is essential for protein transport across the inner membrane of Gram-negative bacteria, yet its role in β-lactamase secretion under antibiotic stress and its therapeutic potential remain unexplored. Here, we demonstrate that the SecYEG complex serves as the primary translocation channel for NDM-1, CTX-M-9, and TEM-1, and that sub-inhibitory imipenem and cefepime exposure coordinately upregulate both β-lactamase genes (blaNDM-1, blaCTX-M-9, blaTEM-1) and SecYEGtranslocon components (secY, secE, secG) in recombinant E. coli models. This transcriptional co-upregulation correlates with increased β-lactamase activity, enhanced hydrolytic capacity, and the emergence of a multidrug-resistant phenotype, revealing a previously unrecognized adaptive mechanism whereby antibiotic stress potentiates resistance by boosting secretory machinery. Importantly, the natural flavonoid luteolin-7-O-glucoside (LOG) effectively reverses this phenotype-downregulating resistance and translocon gene expression, reducing enzymatic activity, and restoring β-lactam susceptibility. Mechanistically, molecular docking and MST confirmed that LOG binds directly to NDM-1 (Gln123, Lys211, and Asn220)and to specific residues (Ile82, Ile278) on SecY.These findings establish the SecYEGtransloconas a viable, stress-responsive therapeutic target and identify LOG as a first-in-class dual-acting adjuvant that inhibits β-lactamase activity and blocks its SecY-dependent translocation, offering a promising strategy to combat multidrug-resistant CRE infections.

Steps to reproduce

1. Strains Host: E. coli BL21(DE3) . Plasmids: pRSFDuet-1-blaNDM-1-blaCTX-M-9 and pRSFDuet-1-blaNDM-1-blaTEM-1. Antibiotics: IPM, FEP, MEM, FOX, CAZ, SCF (National Institute, Beijing). LOG: Luteolin-7-O-glucoside . 2. Plasmid construction Amplified blaNDM-1, blaCTX-M-9, blaTEM-1 from pCYNDM01 (MK510953) with specific primers (SalI/HindIII for blaNDM-1; NdeI/XhoI for blaCTX-M-9 and blaTEM-1). Cloned into pRSFDuet-1 (6×His-tag) via T4 ligase. Verified by sequencing, transformed into BL21(DE3), selected on LB+kanamycin (100 µg/mL) + IPM (0.5 µg/mL). 3. Antibiotic exposure & LOG treatment Stepwise adaptation in MH broth at 37°C, 200 rpm, starting from 0.5×MIC of IPM, FEP, or IPM+FEP. Subculture (1:100) at same concentration until growth time (OD600 1.5–2) stabilized within 5–12 h over 3 passages. Increased by 0.25 µg/mL until target: 1.5 µg/mL (phenotype/genomics) or 48 µg/mL (protein). LOG stepwise (0–32 µg/mL; 32 µg/mL for protein) for 8 h from adapted strains. Three biological replicates. 4. MIC determination (VITEK-2) Tested IPM, MEM, FOX, CAZ, FEP, SCF against strains exposed to 1.5 µg/mL antibiotics ± LOG (0–4 µg/mL). Inoculum: ~1.5×10⁶ CFU/mL in 96-well plates with serial antibiotics (0.5–1024 µg/mL) + LOG, incubated 37°C, 16–24 h. Interpreted by CLSI M100 (2019). Three replicates. 5. qRT-PCR RNA extracted (TIANGEN kit) from strains exposed to 1.5 µg/mL antibiotics + 2 µg/mL LOG. cDNA synthesis (TOYOBO kit). Primers for 16S rRNA, blaNDM-1, blaCTX-M-9, blaTEM-1, secY, secE, secG . 6. SDS-PAGE, Western blot, β-lactamase activity Strains exposed to 48 µg/mL antibiotics + 32 µg/mL LOG. Protein expression: IPTG 0.5 mM, 16°C, 20 h. Purification: His-tag (Ni-NTA) for NDM-1; S-tag (immunoprecipitation) for CTX-M-9/TEM-1. SDS-PAGE (12%), Western blot with anti-NDM-1 (1:1000) and HRP-secondary. Activity: 5 µg purified enzyme + IPM (0–1024 µg/mL) with E. coli BL21(DE3) (~1.5×10⁶ CFU/mL); MIC read at 4, 8, 12, 24 h. 7. Morphology & structure Strain: E. coli BL21(DE3)-blaNDM-1-blaCTX-M-9 exposed to 48 µg/mL IPM + 32 µg/mL LOG. Light microscopy: Gram stain. Fluorescence: PI staining (20 µg/mL, 15 min) for membrane permeability, visualized at 400×. TEM: Fixed in 2.5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated, embedded in epoxy resin, sectioned (60 nm), stained with uranyl acetate and lead citrate; imaged at 7000× (HT7800). 8. Molecular docking LOG docking into SecYEG and NDM-1 using AutoDock 4.1 (300 GA runs). Binding energies calculated for SecY, SecE, SecG. NDM-1 active site defined from ampicillin complex, retaining zinc-bound water. 9. Synthetic peptide Peptides for SecY residues Ile82 (P82) and Ile278 (P278) synthesized by Fmoc SPPS, verified by MS. 10.MST Monolith NT.115. Labeled 0.25 µM P82, P278, or NDM-1 ± 1 mM LOG, 15-stepdilution. KD calculated (MO Affinity v2.3), SNR >5 significant. Also tested peptide–NDM-1 interactions. 11. Statistics Mean ± SD from ≥3 independent experiments. Mann-Whitney U test, P < 0.05 significant .

Institutions

• Hubei University of Medicine

  Hubei, Shiyan

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