skip to main content


Title: Visualizing the Dynamic Metalation State of New Delhi Metallo-β-lactamase-1 in Bacteria Using a Reversible Fluorescent Probe
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.  more » « less
Award ID(s):
1903808
NSF-PAR ID:
10233284
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ;
Date Published:
Journal Name:
Journal of the American Chemical Society
ISSN:
0002-7863
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. null (Ed.)
    The visualization of chloride in living cells with fluorescent sensors is linked to our ability to design hosts that can overcome the energetic penalty of desolvation to bind chloride in water. Fluorescent proteins can be used as biological supramolecular hosts to address this fundamental challenge. Here, we showcase the power of protein engineering to convert the fluorescent proton-pumping rhodopsin GR from Gloeobacter violaceus into GR1, a red-shifted, turn-on fluorescent sensor for chloride in detergent micelles and in live Escherichia coli . This non-natural function was unlocked by mutating D121, which serves as the counterion to the protonated retinylidene Schiff base chromophore. Substitution from aspartate to valine at this position (D121V) creates a binding site for chloride. The binding of chloride tunes the p K a of the chromophore towards the protonated, fluorescent state to generate a pH-dependent response. Moreover, ion pumping assays combined with bulk fluorescence and single-cell fluorescence microscopy experiments with E. coli , expressing a GR1 fusion with a cyan fluorescent protein, show that GR1 does not pump ions nor sense membrane potential but instead provides a reversible, ratiometric readout of changes in extracellular chloride at the membrane. This discovery sets the stage to use natural and laboratory-guided evolution to build a family of rhodopsin-based fluorescent chloride sensors with improved properties for cellular applications and learn how proteins can evolve and adapt to bind anions in water. 
    more » « less
  2. The zebrafish is a powerful model organism to study the mechanisms governing transition metal ions within whole brain tissue. Zinc is one of the most abundant metal ions in the brain, playing a critical pathophysiological role in neurodegenerative diseases. The homeostasis of free, ionic zinc (Zn2+) is a key intersection point in many of these diseases, including Alzheimer’s disease and Parkinson’s disease. A Zn2+ imbalance can eventuate several disturbances that may lead to the development of neurodegenerative changes. Therefore, compact, reliable approaches that allow the optical detection of Zn2+ across the whole brain would contribute to our current understanding of the mechanisms that underlie neurological disease pathology. We developed an engineered fluorescence protein-based nanoprobe that can spatially and temporally resolve Zn2+ in living zebrafish brain tissue. The self-assembled engineered fluorescence protein on gold nanoparticles was shown to be confined to defined locations within the brain tissue, enabling site specific studies, compared to fluorescent protein-based molecular tools, which diffuse throughout the brain tissue. Two-photon excitation microscopy confirmed the physical and photometrical stability of these nanoprobes in living zebrafish (Danio rerio) brain tissue, while the addition of Zn2+ quenched the nanoprobe fluorescence. Combining orthogonal sensing methods with our engineered nanoprobes will enable the study of imbalances in homeostatic Zn2+ regulation. The proposed bionanoprobe system offers a versatile platform to couple metal ion specific linkers and contribute to the understanding of neurological diseases. 
    more » « less
  3. Cell–cell interactions are critical for transmitting signals among cells and maintaining their normal functions from the single-cell level to tissues. In cancer studies, interactions between drug-resistant and drug-sensitive cells play an important role in the development of chemotherapy resistance of tumors. As metabolites directly reflect the cell status, metabolomics studies provide insight into cell–cell communication. Mass spectrometry (MS) is a powerful tool for metabolomics studies, and single cell MS (SCMS) analysis can provide unique information for understanding interactions among heterogeneous cells. In the current study, we utilized a direct co-culture system (with cell–cell contact) to study metabolomics of single cells affected by cell–cell interactions in their living status. A fluorescence microscope was utilized to distinguish these two types of cells for SCMS metabolomics studies using the Single-probe SCMS technique under ambient conditions. Our results show that through interactions with drug-resistant cells, drug-sensitive cancer cells acquired significantly increased drug resistance and exhibited drastically altered metabolites. Further investigation found that the increased drug resistance was associated with multiple metabolism regulations in drug-sensitive cells through co-culture such as the upregulation of sphingomyelins lipids and lactic acid and the downregulation of TCA cycle intermediates. The method allows for direct MS metabolomics studies of individual cells labeled with fluorescent proteins or dyes among heterogeneous populations. 
    more » « less
  4. Fabrication of highly stable, reversible, and efficient portable sensors for the detection of explosives for safety and security is challenging due to the robustness of the currently available detection tools, limiting their mass deployment to the explosion prone areas. This paper reports a new direction towards the sensing of nitro- and peroxide-based explosives using highly stable rare-earth-doped BaWO 4 nanofibers with remarkable sensitivity and reversibility. BaWO 4 nanofibers doped with Tb 3+ and Eu 3+ ions are fabricated through a sol–gel electrospinning process, and their emission characteristics and application as a fluorescent probe for the sensing of 2-nitrotoluene and H 2 O 2 , explosive taggants representing a broad class of explosives, are studied in detail. Scheelite structured BaWO 4 nanofibers exhibit excellent luminescence characteristics, and the rare-earth ion doping in the polycrystalline BaWO 4 nanofibers is tailored to achieve blue, green, red, and white light emissions. These nanofibers are extremely sensitive to 2-nitrotoluene and H 2 O 2 with rapid response time, and sensitivity is observed within the range of 1–400 ppb and 1–10 ppm, towards 2-nitrotoluene and H 2 O 2 , respectively. The fluorescence quenching of BaWO 4 nanofibers in the presence of 2-nitrotoluene and H 2 O 2 is exponential with the quenching constants up to 1.73 × 10 6 and 2.73 × 10 4 L mol −1 , respectively, which are significantly higher than those of most of the fluorescent probes based on metal–organic frameworks and conjugated organic materials. After exposing to 2-nitrotoluene, the luminescence of the nanofibers is retained completely upon heating at 120 °C for 10 min and the sensory response is retained as fresh nanofibers, and currently available fluorescent explosive sensors could not achieve such a recovery. The high sensitivity and selectivity of scalable rare-earth-doped BaWO 4 nanofibers provide a new platform for the simultaneous detection of two classes of explosives. 
    more » « less
  5. In biological systems, chemical and physical transformations of engineered silver nanomaterials (AgENMs) are mediated, in part, by proteins and other biomolecules. Metalloprotein interactions with AgENMs are also central in understanding toxicity, antimicrobial, and resistance mechanisms. Despite their readily available thiolate and amine ligands, zinc finger (ZF) peptides have thus far escaped study in reaction with AgENMs and their Ag( i ) oxidative dissolution product. We report spectroscopic studies that characterize AgENM and Ag( i ) interactions with two ZF peptides that differ in sequence, but not in metal binding ligands: the ZF consensus peptide CP-CCHC and the C-terminal zinc finger domain of HIV-1 nucleocapsid protein p7 (NCp7_C). Both ZF peptides catalyze AgENM (10 and 40 nm, citrate coated) dissolution and agglomeration, two important AgENM transformations that impact bioreactivity. AgENMs and their oxidative dissolution product, Ag( i )(aq), mediate changes to ZF peptide structure and metalation as well. Spectroscopic titrations of Ag( i ) into apo-ZF peptides show an Ag( i )–thiolate charge transfer band, indicative of Ag( i )–ZF binding. Fluorescence studies of the Zn( ii )–NCp_7 complex indicate that the Ag( i ) also effectively competes with the Zn( ii ) to drive Zn( ii ) displacement from the ZFs. Upon interaction with AgENMs, Zn( ii ) bound ZF peptides show a secondary structural change in circular dichroism spectroscopy toward an apo-like structure. The results suggest that Ag( i ) and AgENMs may alter ZF protein function within the cell. 
    more » « less