Direct assessment of nitrative stress in lipid environments: Applications of a designer lipid-based biosensor for peroxynitrite.

Lipid membranes and lipid-rich organelles are targets of peroxynitrite (ONOO-), a highly reactive species generated under nitrative stress. We report a membrane-localized phospholipid (DPPC-TC-ONOO-) that allows the detection of ONOO- in diverse lipid environments: biomimetic vesicles, mammalian cell compartments, and within the lung lining. DPPC-TC-ONOO- and POPC self-assemble to membrane vesicles that fluorogenically and selectively respond to ONOO-. DPPC-TC-ONOO-, delivered through lipid nanoparticles, allowed for ONOO- detection in the endoplasmic reticulum upon cytokine-induced nitrative stress in live mammalian cells. It also responded to ONOO- within lung tissue murine models upon acute lung injury. We observed nitrative stress around bronchioles in precision cut lung slices exposed to nitrogen mustard and in pulmonary macrophages following intratracheal bleomycin challenge. Results showed that DPPC-TC-ONOO- functions specifically toward iNOS, a key enzyme modulating nitrative stress, and offers significant advantages over its hydrophilic analog in terms of localization and signal generation.

Synthesis of vancomycin fluorescent probes that retain antimicrobial activity, identify Gram-positive bacteria, and detect Gram-negative outer membrane damage.

Antimicrobial resistance is an urgent threat to human health, and new antibacterial drugs are desperately needed, as are research tools to aid in their discovery and development. Vancomycin is a glycopeptide antibiotic that is widely used for the treatment of Gram-positive infections, such as life-threatening systemic diseases caused by methicillin-resistant Staphylococcus aureus (MRSA). Here we demonstrate that modification of vancomycin by introduction of an azide substituent provides a versatile intermediate that can undergo copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction with various alkynes to readily prepare vancomycin fluorescent probes. We describe the facile synthesis of three probes that retain similar antibacterial profiles to the parent vancomycin antibiotic. We demonstrate the versatility of these probes for the detection and visualisation of Gram-positive bacteria by a range of methods, including plate reader quantification, flow cytometry analysis, high-resolution microscopy imaging, and single cell microfluidics analysis. In parallel, we demonstrate their utility in measuring outer-membrane permeabilisation of Gram-negative bacteria. The probes are useful tools that may facilitate detection of infections and development of new antibiotics.

Bioorthogonal Functionalization of Material Surfaces with Bioactive Molecules.

The functionalization of material surfaces with biologically active molecules is crucial for enabling technologies in life sciences, biotechnology, and medicine. However, achieving biocompatibility and bioorthogonality with current synthetic methods remains a challenge. We report herein a novel surface functionalization method that proceeds chemoselectively and without a free transition metal catalyst. In this method, a coating is first formed via the tyrosinase-catalyzed putative polymerization of a tetrazine-containing catecholamine (DOPA-Tet). One or more types of molecule of interest containing trans-cyclooctene are then grafted onto the coating via tetrazine ligation. The entire process proceeds under physiological conditions and is suitable for grafting bioactive molecules with diverse functions and structural complexities. Utilizing this method, we functionalized material surfaces with enzymes (alkaline phosphatase, glucose oxidase, and horseradish peroxidase), a cyclic peptide (cyclo[Arg-Gly-Asp-D-Phe-Lys], or c(RGDfK)), and an antibiotic (vancomycin). Colorimetric assays confirmed the maintenance of the biocatalytic activities of the grafted enzymes on the surface. We established the mammalian cytocompatibility of the functionalized materials with fibroblasts. Surface functionalization with c(RGDfK) showed improved fibroblast cell morphology and cytoskeletal organization. Microbiological studies with Staphylococcus aureus indicated that surfaces coated using DOPA-Tet inhibit the formation of biofilms. Vancomycin-grafted surfaces additionally display significant inhibition of planktonic S. aureus growth.

Fast bacterial growth reduces antibiotic accumulation and efficacy.

Phenotypic variations between individual microbial cells play a key role in the resistance of microbial pathogens to pharmacotherapies. Nevertheless, little is known about cell individuality in antibiotic accumulation. Here, we hypothesise that phenotypic diversification can be driven by fundamental cell-to-cell differences in drug transport rates. To test this hypothesis, we employed microfluidics-based single-cell microscopy, libraries of fluorescent antibiotic probes and mathematical modelling. This approach allowed us to rapidly identify phenotypic variants that avoid antibiotic accumulation within populations of Escherichia coli, Pseudomonas aeruginosa, Burkholderia cenocepacia, and Staphylococcus aureus. Crucially, we found that fast growing phenotypic variants avoid macrolide accumulation and survive treatment without genetic mutations. These findings are in contrast with the current consensus that cellular dormancy and slow metabolism underlie bacterial survival to antibiotics. Our results also show that fast growing variants display significantly higher expression of ribosomal promoters before drug treatment compared to slow growing variants. Drug-free active ribosomes facilitate essential cellular processes in these fast-growing variants, including efflux that can reduce macrolide accumulation. We used this new knowledge to eradicate variants that displayed low antibiotic accumulation through the chemical manipulation of their outer membrane inspiring new avenues to overcome current antibiotic treatment failures.

Bacteria can cause an array of diseases ranging from mildly inconvenient to deadly. In fact, every year around the world, five million people succumb to a bacterial infection. Antibiotics can kill bacteria or stop their growth, but many bacterial species are now able to evade these drugs. To be efficient, most antibiotics first need to get inside a bacterium; there, they accumulate until they reach the concentration they need to act. Often, the drugs make their way through channel-like structures ('pores') studded through the external membranes of bacteria and which control the passage of molecules in and out of cells. Resistance usually emerges when genetic changes provide the microorganism with an advantage against antibiotics, or when the microorganism performs the biochemical reactions necessary for life at a slower pace. In contrast, Lapinska, Pagliara et al. decided to examine how genetically similar Escherichia coli bacteria which differed in their growth rate would fare against antibiotics. The drug targeted ribosomes, the machinery that produces proteins in a cell. A combination of techniques was used to follow individual cells, revealing that fast-growing variants better managed to survive. A closer look showed that bacteria which were growing quickly had a surplus of ribosomes, which then produced more pores that could pump the antibiotic out the cell. Next, Lapinska, Pagliara et al. exposed the bacteria to both the antibiotic and a compound that weakens bacterial membrane; this erased the advantage shown by the fast-growing variants. Overall, this work gives a finer understanding of the mechanisms that underlie antibiotic resistance, which could help pave the way to new strategies to combat harmful bacteria.

Application of antibiotic-derived fluorescent probes to bacterial studies.

Fluorescent probes are extensively applied as useful tools for imaging and determining dynamic processes in bacterial cells. In particular, antibiotic-derived fluorescent probes which can visualize the presence or the localization of antibiotics within bacteria through the monitoring of changes in fluorescence signal, are particularly useful. They form an emerging set of tools for studying the mode of action of their parent antibiotics and examining bacterial resistance and persistence, with the long-term goal of developing fresh approaches to the treatment of drug-resistant bacterial infections. In this chapter, we discuss the applications of antibiotic-based fluorescent probes to visualize bacteria, focusing on the techniques we have utilized to study their localization, penetration and efflux. We describe detailed protocols for analysis of bacteria using microscopy, flow cytometry, and plate reader-based methods based on these probes.

CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage.

The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.

Serum Complement Activation by C4BP-IgM Fusion Protein Can Restore Susceptibility to Antibiotics in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is the etiological agent of gonorrhea, the second most common bacterial sexually transmitted infection worldwide. Reproductive sequelae of gonorrhea include infertility, ectopic pregnancy and chronic pelvic pain. Most antibiotics currently in clinical use have been rendered ineffective due to the rapid spread of antimicrobial resistance among gonococci. The developmental pipeline of new antibiotics is sparse and novel therapeutic approaches are urgently needed. Previously, we utilized the ability of N. gonorrhoeae to bind the complement inhibitor C4b-binding protein (C4BP) to evade killing by human complement to design a chimeric protein that linked the two N-terminal gonococcal binding domains of C4BP with the Fc domain of IgM. The resulting molecule, C4BP-IgM, enhanced complement-mediated killing of gonococci. Here we show that C4BP-IgM induced membrane perturbation through complement deposition and membrane attack complex pore insertion facilitates the access of antibiotics to their intracellular targets. Consequently, bacteria become more susceptible to killing by antibiotics. Remarkably, C4BP-IgM restored susceptibility to azithromycin of two azithromycin-resistant clinical gonococcal strains because of overexpression of the MtrC-MtrD-MtrE efflux pump. Our data show that complement activation can potentiate activity of antibiotics and suggest a role for C4BP-IgM as an adjuvant for antibiotic treatment of drug-resistant gonorrhea.

Two for the Price of One: Heterobivalent Ligand Design Targeting Two Binding Sites on Voltage-Gated Sodium Channels Slows Ligand Dissociation and Enhances Potency.

Voltage-gated sodium (NaV) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of NaV channel ligands. We conjugated µ-conotoxin KIIIA, which occludes the pore of the NaV channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide-alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40-120 Å. The ligand with an 80 Å linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4-24-fold higher potency compared to those of the monovalent peptides for the human NaV1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of NaV channels.

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescent macrolide probes - synthesis and use in evaluation of bacterial resistance.

The emerging crisis of antibiotic resistance requires a multi-pronged approach in order to avert the onset of a post-antibiotic age. Studies of antibiotic uptake and localisation in live cells may inform the design of improved drugs and help develop a better understanding of bacterial resistance and persistence. To facilitate this research, we have synthesised fluorescent derivatives of the macrolide antibiotic erythromycin. These analogues exhibit a similar spectrum of antibiotic activity to the parent drug and are capable of labelling both Gram-positive and -negative bacteria for microscopy. The probes localise intracellularly, with uptake in Gram-negative bacteria dependent on the level of efflux pump activity. A plate-based assay established to quantify bacterial labelling and localisation demonstrated that the probes were taken up by both susceptible and resistant bacteria. Significant intra-strain and -species differences were observed in these preliminary studies. In order to examine uptake in real-time, the probe was used in single-cell microfluidic microscopy, revealing previously unseen heterogeneity of uptake in populations of susceptible bacteria. These studies illustrate the potential of fluorescent macrolide probes to characterise and explore drug uptake and efflux in bacteria.

Non-antibiotic Small-Molecule Regulation of DHFR-Based Destabilizing Domains In Vivo.

The E. coli dihydrofolate reductase (DHFR) destabilizing domain (DD), which shows promise as a biologic tool and potential gene therapy approach, can be utilized to achieve spatial and temporal control of protein abundance in vivo simply by administration of its stabilizing ligand, the routinely prescribed antibiotic trimethoprim (TMP). However, chronic TMP use drives development of antibiotic resistance (increasing likelihood of subsequent infections) and disrupts the gut microbiota (linked to autoimmune and neurodegenerative diseases), tempering translational excitement of this approach in model systems and for treating human diseases. Herein, we identified a TMP-based, non-antibiotic small molecule, termed 14a (MCC8529), and tested its ability to control multiple DHFR-based reporters and signaling proteins. We found that 14a is non-toxic and can effectively stabilize DHFR DDs expressed in mammalian cells. Furthermore, 14a crosses the blood-retinal barrier and stabilizes DHFR DDs expressed in the mouse eye with kinetics comparable to that of TMP (≤6 h). Surprisingly, 14a stabilized a DHFR DD in the liver significantly better than TMP did, while having no effect on the mouse gut microbiota. Our results suggest that alternative small-molecule DHFR DD stabilizers (such as 14a) may be ideal substitutes for TMP in instances when conditional, non-antibiotic control of protein abundance is desired in the eye and beyond.

Fluoroquinolone-derived fluorescent probes for studies of bacterial penetration and efflux.

Fluorescent probes derived from the fluoroquinolone antibiotic ciprofloxacin were synthesised using a Cu(i)-catalysed azide-alkyne cycloaddition (CuAAC) to link a ciprofloxacin azide derivative with alkyne-substituted green and blue fluorophores. The azide (2) and fluorophore (3 and 4) derivatives retained antimicrobial activity against Gram-positive and Gram-negative bacteria. The use of confocal fluorescent microscopy showed intracellular penetration, which was substantially enhanced in the presence of carbonyl cyanide 3-chlorophenylhydrazone as an efflux pump inhibitor in Escherichia coli.

Fluorescent Antibiotics: New Research Tools to Fight Antibiotic Resistance.

Better understanding how multidrug-resistant (MDR) bacteria can evade current and novel antibiotics requires a better understanding of the chemical biology of antibiotic action. This necessitates using new tools and techniques to advance our knowledge of bacterial responses to antibiotics, ideally in live cells in real time, to selectively investigate bacterial growth, division, metabolism, and resistance in response to antibiotic challenge. In this review, we discuss the preparation and biological evaluation of fluorescent antibiotics, focussing on how these reporters and assay methods can help elucidate resistance mechanisms. We also examine the potential utility of such probes for real-time in vivo diagnosis of infections.