Two-Pronged Dormant Photosensitizer-Antibiotic Bacterial Inactivation: Mechanism, Dosage, and Cellular Evolution Visualized at the Single-Cell Level.

Innovative therapeutic approaches are required to battle the rise of antibiotic-resistant bacterial strains. Tapping on reactive oxygen species (ROS) generation in bacteria induced by bactericidal antibiotics, here we report a two-pronged strategy for bacterial inactivation relying on the synergistic combination of a bactericidal antibiotic and newly designed dormant photosensitizers (DoPSs) that activate in the presence of ROS. Intramolecular quenching renders DoPS inert in the presence of light. ROS trapping by DoPS aborts the quenching mechanism unmasking, in equal proportions, singlet oxygen (1O2) sensitization and fluorescence emission. Juxtaposed antioxidant-prooxidant activity built within our DoPS enables (i) initial activation of a few molecules by ROS and (ii) subsequent rapid activation of all DoPS in a bacterium via a domino effect mediated by photogenerated 1O2. Bulk colony forming unit studies employing the minimum inhibitory concentration of the antibiotic illustrate rapid and selective inactivation of Escherichia coli and Pseudomonas aeruginosa only in the presence of light, antibiotic, and DoPS. Single-cell, real-time imaging studies on E. coli reveal an autocatalytic progression of DoPS activation from focal points, providing a unique amplification system for sensing. Single-cell analysis further illustrates the impact of DoPS cellular loading on the rate of DoPS activation and cell death times and on the 1O2 dosing necessary for cell death to occur. Our two-pronged therapy discriminates based on cell metabolites and has the potential to result in lower toxicity, pave the way to reduced drug resistance, and provide insightful mechanistic information about bacterial membrane response to 1O2.

Binding and Sliding Dynamics of the Hepatitis C Virus Polymerase: Hunting the 3' Terminus.

The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase catalyzes the replication of the (+) single-stranded RNA genome of HCV. In vitro studies have shown that replication can be performed in the absence of a primer. However, the dynamics and mechanism by which NS5B locates the 3'-terminus of the RNA template to initiate de novo synthesis remain elusive. Here, we performed single-molecule fluorescence studies based on protein-induced fluorescence enhancement reporting on NS5B dynamics on a short model RNA substrate. Our results suggest that NS5B exists in a fully open conformation in solution wherefrom it accesses its binding site along RNA and then closes. Our results revealed two NS5B binding modes: an unstable one resulting in rapid dissociation, and a stable one characterized by a larger residence time on the substrate. We associate these bindings to an unproductive and productive orientation, respectively. Addition of extra mono (Na+)- and divalent (Mg2+) ions increases the mobility of NS5B along its RNA substrate. However, only Mg2+ ions induce a decrease in NS5B residence time. Dwell times of residence increase with the length of the single-stranded template, suggesting that NS5B unbinds its substrate by unthreading the template rather than by spontaneous opening.

Alkylation converts riboflavin into an efficient photosensitizer of phospholipid membranes.

A new decyl chain [-(CH2)9CH3] riboflavin conjugate has been synthesized and investigated. A nucleophilic substitution (SN2) reaction was used for coupling the alkyl chain to riboflavin (Rf), a model natural photosensitizer. As expected, the alkylated compound (decyl-Rf) is significantly more lipophilic than its precursor and efficiently intercalates within phospholipid bilayers, increasing its fluorescence quantum yield. The oxidative damage to lipid membranes photoinduced by decyl-Rf was investigated in large and giant unilamellar vesicles (LUVs and GUVs, respectively) composed of different phospholipids. Using a fluorogenic probe, fast radical formation and singlet oxygen generation was demonstrated upon UVA irradiation in vesicles containing decyl-Rf. Photosensitized formation of conjugated dienes and hydroperoxides, and membrane leakage in LUVs rich in poly-unsaturated fatty acids were also investigated. The overall assessment of the results shows that decyl-Rf is a significantly more efficient photosensitizer of lipids than its unsubstituted precursor and that the association to lipid membranes is key to trigger phospholipid oxidation. Alkylation of hydrophilic photosensitizers as a simple and general synthetic tool to obtain efficient photosensitizers of biomembranes, with potential applications, is discussed.

Singlet Oxygen Flux, Associated Lipid Photooxidation, and Membrane Expansion Dynamics Visualized on Giant Unilamellar Vesicles.

The physical properties of lipid membranes depend on their lipid composition. Photosensitized singlet oxygen (1O2) provides a handle to spatiotemporally control the generation of lipid hydroperoxides via the ene reaction, enabling fundamental studies on membrane dynamics in response to chemical composition changes. Critical to relating the physical properties of the lipid membrane to hydroperoxide formation is the availability of a sensitive reporter to quantify the arrival of 1O2. Here, we show that a fluorogenic α-tocopherol analogue, H4BPMHC, undergoes a >360-fold emission intensity enhancement in liposomes following a reaction with 1O2. Rapid quenching of 1O2 by the probe (kq = 4.9 × 108 M-1 s-1) ensures zero-order kinetics of probe consumption. The remarkable intensity enhancement of H4BPMHC upon 1O2 trapping, its linear temporal behavior, and its protective role in outcompeting membrane damage provide a sensitive and reliable method to quantify the 1O2 flux on lipid membranes. Armed with this probe, fluorescence microscopy studies were devised to enable (i) monitoring the flux of photosensitized 1O2 into giant unilamellar vesicles (GUVs), (ii) establishing the onset of the ene reaction with the double bonds of monounsaturated lipids, and (iii) visualizing the ensuing collective membrane expansion dynamics associated with molecular changes in the lipid structure upon hydroperoxide formation. A correlation was observed between the time for antioxidant H4BPMHC consumption by 1O2 and the onset of membrane fluctuations and surface expansion. Together, our imaging studies with H4BPMHC in GUVs provide a methodology to explore the intimate relationship between photosensitizer activity, chemical insult, membrane morphology, and its collective dynamics.

Fluorescence-Amplified Detection of Redox Turnovers in Supported Lipid Bilayers Illuminates Redox Processes of α-Tocopherol.

Electron-transfer processes in lipid membranes are key to biological functions, yet challenging to study because of the intrinsic heterogeneity of the systems. Here, we report spectro-electrochemical measurements on indium tin oxide-supported lipid bilayers toward the selective induction and sensing of redox processes in membranes. Working at neutral pH with a fluorogenic α-tocopherol analogue, the dynamics of the two-electron oxidation of the chromanol to a chromanone and the rapid thermal decay of the latter to a chromoquinone are recorded as a rapid surge and drop in intensity, respectively. Continuous voltage cycling reveals rapid chromoquinone two-electron, two-proton reduction to dihydrochromoquinone at negative bias, followed by slow regeneration of the former at positive bias. The kinetic parameters of these different transitions are readily obtained as a function of applied potentials. The sensitivity and selectivity afforded by the reported method enables monitoring signals equivalent to femtoampere currents with a high signal-to-background ratio. The study provides a new method to monitor membrane redox processes with high sensitivity and minimal concentrations and unravels key dynamic aspects of α-tocopherol redox chemistry.

Chemically Tuned, Reversible Fluorogenic Electrophile for Live Cell Nanoscopy.

We report a chemically tuned fluorogenic electrophile designed to conduct live-cell super-resolution imaging by exploiting its stochastic reversible alkylation reaction with cellular nucleophiles. Consisting of a lipophilic BODIPY fluorophore tethered to an electrophilic cyanoacrylate warhead, the new probe cyanoAcroB remains nonemissive due to internal conversion along the cyanoacrylate moiety. Intermittent fluorescence occurs following thiolate Michael addition to the probe, followed by retro-Michael reaction, tuned by the cyano moiety in the acrylate warhead and BODIPY decoration. This design enables long-term super-resolved imaging of live cells by preventing fluorescent product accumulation and background increase, while preserving the pool of the probe. We demonstrate the imaging capabilities of cyanoAcroB via two methods: (i) single-molecule localization microscopy imaging with nanometer accuracy by stochastic chemical activation and (ii) super-resolution radial fluctuation. The latter tolerates higher probe concentrations and low imaging powers, as it exploits the stochastic adduct dissociation. Super-resolved imaging with cyanoAcroB reveals that electrophile alkylation is prevalent in mitochondria and endoplasmic reticulum. The 2D dynamics of these organelles within a single cell are unraveled with tens of nanometers spatial and sub-second temporal resolution through continuous imaging of cyanoAcroB extending for tens of minutes. Our work underscores the opportunities that reversible fluorogenic probes with bioinspired warheads bring toward illuminating chemical reactions with super-resolved features in live cells.

Postmetalated Zirconium Metal Organic Frameworks as a Highly Potent Bactericide.

Metal-organic frameworks (MOFs) have emerged as an important class of hybrid organic-inorganic materials. One of the reasons they have gained remarkable attention is attributed to the possibility of altering them by postsynthetic modification, thereby providing access to new and novel advanced materials. MOFs have been applied in catalysis, gas storage, gas separation, chemical sensing, and drug delivery. However, their bactericidal use has rarely been explored. Herein, we developed a two-step process for the synthesis of zirconium-based MOFs metalated with silver cations as a potent antibacterial agent. The obtained products were thoroughly characterized by powder X-ray diffraction, scanning electron microscopy, UV-visible, IR, thermogravimetric, and Brunauer-Emmett-Teller analyses. Their potency was evaluated against E. coli with a reported minimal inhibitory concentration and minimal bactericidal concentration of as low as 6.5 µg/mL of silver content. Besides the novelty of the system, the advantage of this strategy is that the MOFs could be potentially regenerated and remetalated after each antibacterial test, unlike previously reported frameworks, which involved the destruction of the framework.