Visualizing the Dynamic Metalation State of New Delhi Metallo-β-lactamase-1 in Bacteria Using a Reversible Fluorescent Probe

Abstract

New Delhi metallo-β-lactamase (NDM) grants resistance to a broad spectrum of β-lactam antibiotics, including last-resort carbapenems, and is emerging as a global antibiotic resistance threat. Limited zinc availability adversely impacts the ability of NDM-1 to provide resistance, but a number of clinical variants have emerged that are more resistant to zinc scarcity (e.g., NDM-15). To provide a novel tool to better study metal ion sequestration in host-pathogen interactions, we describe the development of a fluorescent probe that reports on the dynamic metalation state of NDM within Escherichia coli. The thiol-containing probe selectively coordinates the dizinc metal cluster of NDM and results in a 17-fold increase in fluorescence intensity. Reversible binding enables competition and time-dependent studies that reveal fluorescence changes used to detect enzyme localization, substrate and inhibitor engagement, and changes to metalation state through the imaging of live E. coli using confocal microscopy. NDM-1 is shown to be susceptible to demetalation by intracellular and extracellular metal chelators in a live-cell model of zinc dyshomeostasis, whereas the NDM-15 metalation state is shown to be more resistant to zinc flux. The development of this reversible turn-on fluorescent probe for the metalation state of NDM provides a new tool for monitoring the impact of metal ion sequestration by host defense mechanisms and for detecting inhibitor-target engagement during the development of therapeutics to counter this resistance determinant.

Figure 1.

(A) Design of reversible NDM-1 fluorescent probes. (B) Structures of probes 1–4. (C) Fluorescence fold turn-on of probes with NDM-1 (1:3 ratio, 10 μM probe; λ_ex = 420 nm). (D) Fluorescence spectra showing the fluorescence turn-on for probe 4D (10 μM) with increasing equivalents of NDM-1. λ_ex = 420 nm. (E) Probes showing the best fluorescence response with NDM-1. All studies were conducted in degassed 50 mM HEPES, 10 μM ZnSO₄ buffer, pH 7.0, at room temperature using acetonitrile (≤5% v/v) as a cosolvent.

Figure 2.

Proposed binding modes from QM/DMD simulations for probe 4D with NDM-1 (PDB: 4EXS), with insets showing (A) interactions between the fluorophore end of the probe and hydrophobic M67 in Loop 3. (B) Interaction between N220 and the carbonyl oxygen of the imide ring of the fluorophore. (C) Interaction between K211 and the carbonyl groups in the metal binding group end of the probe.

Figure 3.

(A) Schematic showing the effects of chelator and inhibitor treatments on 4D/NDM-1 mixtures. (B) Change in fluorescence turn-on of 4D-NDM-1 (1:3, 10 μM probe) upon treatments with ZnSO₄ TPEN, dl-captopril (dl-Cap), and dipicolinic acid (DPA). (C) Fluorescence turn-on for 4D with other proteins (1:3, 10 μM probe), human carbonic anhydrase II (hCAII), bovine carbonic anhydrase II (bCAII), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), alkaline phosphatase (AKP), phosphotriesterase (PTE), myoglobin (Mb), and bovine serum albumin (BSA). (D) Fluorescence turn-on for 4D with different NDM-1 isoforms and two other metallo-β-lactamases, VIM-2 and IMP-1 (1:3, 10 μM probe), in the presence of 10 μM ZnSO₄ (light gray) and 50 μM ZnSO₄ (dark gray).

Figure 4.

(A) Confocal fluorescence images of 4D-treated BL21 cells with the Δ35 construct in the presence and absence of IPTG for NDM-1 expression. (B) In-gel fluorescence (probe 4D) and Coomassie staining of native SDS-PAGE run at 120 V for 40 min (4–20% gel) with lysates of BL21 (DE3) cells with the Δ35 construct with and without NDM-1 expression. (C) Confocal fluorescence images of BL21 expressing different NDM-1 constructs (Δ35, FL, C26A) stained with 4D (10 μM). For all imaging experiments, cells were grown in LB broth at 37 °C, supplemented with 0.5 mM IPTG and 50 μM ZnSO₄, and protein expression was induced for 2 h. Prior to imaging, the cells were re-suspended in M9 minimal media to obtain a final OD of ~0.3 for imaging. Scale: 10 μm. λ_ex/λ_em: 405/486–614.

Figure 5.

(A) Schematic showing probe displacement by the substrate and the structure of cephalexin. (B) Time-dependent fluorescence intensity after addition of cephalexin (1 mM) to 4D-treated E. coli BL21 (DE3) expressing Δ35 NDM-1. (C) Example images from 3 min versus 10 min samples used to construct panel B. Effects of dl-captopril (panel D), DPA (panel E), TPEN (panel F), and CaEDTA (panel G) on the fluorescence intensity of BL21 (DE3) cells expressing Δ35 NDM-1 after 20 min incubation (5 min with 8 μM 4D, followed by 15 min of treatment with indicated additives). All data were recorded in triplicate and analyzed using two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Scale bar: 10 μm. λ_ex/λ_em: 405/486–614. All statistical analyses are indicating statistics with respect to the 0 μM treatment.

Figure 6.

(A) Fluorescence image showing expression and localization of NDM-15 cellular Δ35 construct with probe 4D. (B) Effect of addition of TPEN and DPA to NDM-15 Δ35 BL21 cells. All data were recorded in triplicate (2–3 different trials), and two-way ANOVA was performed to determine significance (****p < 0.0001). Scale bar: 10 μm. λ_em/λ_em: 405/486–614. All statistical analyses are indicating statistics with respect to the 0 μM treatment.