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 metallation state of NDM within E. 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 metallation state through the imaging of live E. coli using confocal microscopy. NDM-1 is shown to be susceptible to demetallation by intracellular and extracellular metal chelators in a live-cell model of zinc dyshomeostasis, whereas the NDM-15 metallation state is shown to be more resistant to zinc flux. The development of this reversible turn-on fluorescent probe for the metallation state of NDM provides a new tool for monitoring the impact of metal ion sequestration by host defense mechanisms and to detect inhibitor target engagement during the development of therapeutics to counter this resistance determinant.

Structure and Mechanism-Guided Design of Dual Serine/Metallo-Carbapenemase Inhibitors

“…The horizontal transfer of plasmid-mediated resistance determinants has become a severe threat to the clinical efficacy of antibiotics, in hospital and community settings, triggering the onset of a worldwide antimicrobial resistance crisis. ,, Among them, subclass B1 MβLs including the clinically important NDM-, IMP-, and VIM-type variants and class A KPC-type and class D OXA-type serine carbapenemases are mostly encoded on plasmids in clinical isolates of Acinetobacter spp ., Pseudomonas spp., and Enterobacteriaceae , giving rise to the emergence and spread of MDR Gram-negative pathogens, which threaten the ability to effectively treat hospital-acquired infections. − Global antibiotic resistance has reached critical levels, with major bacterial pathogens, including Gram-negative organisms such as A. baumannii , P. aeruginosa , E. coli , and K. pneumoniae , quickly evolving toward pan-drug-resistant phenotypes. , There is an urgent medical need for strategies that address this carbapenem resistance. , …”

“…32−34 Global antibiotic resistance has reached critical levels, with major bacterial pathogens, including Gram-negative organisms such as A. baumannii, P. aeruginosa, E. coli, and K. pneumoniae, quickly evolving toward pan-drug-resistant phenotypes. 35,36 There is an urgent medical need for strategies that address this carbapenem resistance. 37,38 β-Lactam antibiotics in combination with β-lactamase inhibitors have been demonstrated as a clinically effective strategy to overcome β-lactamase-mediated resistance.…”

Binding of Dual-Function Hybridized Metal–Organic Capsules to Enzymes for Cascade Catalysis

“…17,69 An enhanced luminescence of Pd−ZPP(Fe) (10.0 μM) appeared at both 648 and 714 nm upon the addition of FDH (total 0.5 μM) (Figure 4c). 70 Notably, this spectra revealed significant changes from an initial linear growth to almost unchanged (Figure S27), which could be interpreted as the steady binding between Pd−ZPP(Fe) and enzyme FDH arising from recognition between them. 18,71 Importantly, circular dichroism and luminescence spectra showed that the characteristic peaks attributable to FDH were basically maintained after adding the capsule Pd−ZPP(Fe) and prolonging time, suggesting the stability of the combined system with the secondary and tertiary structures of FDH preserved (Figures S30−S32).…”

Recent Progress in Identifying Bacteria with Fluorescent Probes

“…The conformation of the probe was changed by AmpC, which led to the phenomenon of AIE [ 102 ]. Mehta et al conjugated an environmentally sensitive fluorophore onto a mercaptan-based scaffold of New Delhi metallo- β -lactamase (NDM) inhibitors (probe 35 ) to monitor the dynamic metalation of NDM in E. coli and for fluorescence imaging [ 103 ].…”