Red-Shifted Environmental Fluorophores and Their Use for the Detection of Gram-Negative Bacteria

Two novel, water-soluble, merocyanine fluorophores were readily prepared by microwave-assisted synthesis. Full optical characterization was performed in a series of protic and aprotic solvents, and the dyes displayed fluorescence in the red region with up to a 20-fold decrease in brightness in water, demonstrating a strong environmental sensitivity hereby termed as solvato-fluorogenicity (to distinguish from solvatochromism). Shorter fluorescent lifetimes were also measured in water, which confirmed this character. These dyes were conjugated to a modified polymyxin scaffold that allowed fluorescence “switch-on” upon binding to Gram-negative bacterial membranes, and selective fluorescence detection of bacteria in a wash-free protocol

Self-assembling dipeptide antibacterial nanostructures with membrane disrupting activity.

Peptide-based supramolecular assemblies are a promising class of nanomaterials with important biomedical applications, specifically in drug delivery and tissue regeneration. However, the intrinsic antibacterial capabilities of these assemblies have been largely overlooked. The recent identification of common characteristics shared by antibacterial and self-assembling peptides provides a paradigm shift towards development of antibacterial agents. Here we present the antibacterial activity of self-assembled diphenylalanine, which emerges as the minimal model for antibacterial supramolecular polymers. The diphenylalanine nano-assemblies completely inhibit bacterial growth, trigger upregulation of stress-response regulons, induce substantial disruption to bacterial morphology, and cause membrane permeation and depolarization. We demonstrate the specificity of these membrane interactions and the development of antibacterial materials by integration of the peptide assemblies into tissue scaffolds. This study provides important insights into the significance of the interplay between self-assembly and antimicrobial activity and establishes innovative design principles toward the development of antimicrobial agents and materials

The influence of N-acetyl-L-cysteine on oxidative stress and nitric oxide synthesis in stimulated macrophages treated with a mustard gas analogue

Background: Sulphur mustard gas, 2, 2′-dichlorodiethyl sulphide (HD), is a chemical warfare agent. Both mustard gas and its monofunctional analogue, 2-chloroethyl ethyl sulphide (CEES), are alkylating agents that react with and diminish cellular thiols and are highly toxic. Previously, we reported that lipopolysaccharide (LPS) significantly enhances the cytotoxicity of CEES in murine RAW 264.7 macrophages and that CEES transiently inhibits nitric oxide (NO) production via suppression of inducible NO synthase (iNOS) protein expression. NO generation is an important factor in wound healing. In this paper, we explored the hypotheses that LPS increases CEES toxicity by increasing oxidative stress and that treatment with N-acetyl-L-cysteine (NAC) would block LPS induced oxidative stress and protect against loss of NO production. NAC stimulates glutathione (GSH) synthesis and also acts directly as a free radical scavenger. The potential therapeutic use of the antibiotic, polymyxin B, was also evaluated since it binds to LPS and could thereby block the enhancement of CEES toxicity by LPS and also inhibit the secondary infections characteristic of HD/CEES wounds. Results: We found that 10 mM NAC, when administered simultaneously or prior to treatment with 500 μM CEES, increased the viability of LPS stimulated macrophages. Surprisingly, NAC failed to protect LPS stimulated macrophages from CEES induced loss of NO production. Macrophages treated with both LPS and CEES show increased oxidative stress parameters (cellular thiol depletion and increased protein carbonyl levels). NAC effectively protected RAW 264.7 cells simultaneously treated with CEES and LPS from GSH loss and oxidative stress. Polymyxin B was found to partially block nitric oxide production and diminish CEES toxicity in LPS-treated macrophages. Conclusion: The present study shows that oxidative stress is an important mechanism contributing to CEES toxicity in LPS stimulated macrophages and supports the notion that antioxidants could play a therapeutic role in preventing mustard gas toxicity. Although NAC reduced oxidative stress in LPS stimulated macrophages treated with CEES, it did not reverse CEES-induced loss of NO production. NAC and polymyxin B were found to help prevent CEES toxicity in LPS-treated macrophages

Development And Applications Of Gold-Silica Nanohybrids For Bioanalysis

In the first project, both non-spherical and spherical silica nanomateirals with hollow interiors were synthesized by a one-step soft-templating method. By simply changing the applied solvent from ethanol to 1-propanol, or 1-pentanol, three different shapes of nanomaterials, including nanosphere, nanowire, and nanotadpole, could be obtained, respectively. The effects of other factors on morphology were also systematically studied to propose a growth mechanism. It was found that the PVP-water droplet was the crucial factor on the formation of hollow interiors. Without the addition of PVP, only solid silica nanomaterials, such as solid nanoparticles, nanorods, and nanowires, were synthesized. This developed method showed excellent reproducibility and great potential for a large-scale synthesis. In the second project, a novel nanocomposite contained a spherical gold nanoparticle core, a silica spacer and a fluorescent dye layer in the silica matrix was designed to study the metal-enhanced fluorescence phenomenon. It was found that the size of gold nanoparticles cores and the distances between cores and fluorescent dyes had significant effects on the emitted fluorescence intensity. An enhancement factor of 9.2 was obtained when the nanocomposite contained a 13.7 ± 1.3 nm gold nanoparticle core and a 36.6 ± 4.4 nm silica spacer. In the third project, an ultrasensitive protein assay was developed by using silica nanorods decorated with gold nanoparticles (AuNPs-SiNPs) as labelsin a lateral flow platform. A large number of AuNPs on one single SiNR provided visualized dark purple color that was much darker than the pure AuNPs solution. Therefore, the detection limit was lowered 50 times compared to the traditional AuNP-based lateral flow assay. Under optimal conditions, a linear range of 0.05 - 2 ng/mL with a detection limit of 0.01 ng/mL was obtained. The lateral flow bioassay based on these composite nanomaterials thus offered an ultrasensitive method for rapid detection of trace amount of proteins and has a potential application for point-of-care screening in clinical diagnostics and biomedical research. In the fourth project, a new hyperthermia agent, Au-silica nanowire nanohybrid (Au-SiNW nanohybrid)was synthesized and applied in the photothermal therapy. Due to its strong NIR absorption ability, the Au-SiNW nanohybrid can generate significant amount of heat upon NIR irradiation and induced thermal cell death. By incubating the nanohybrids with cancer cells and irradiating with NIR laser, cancer cells were successfully killed, indicating their potential as promising hyperthermia agents

Dismantling the bacterial glycocalyx: Chemical tools to probe, perturb, and image bacterial glycans

The bacterial glycocalyx is a quintessential drug target comprised of structurally distinct glycans. Bacterial glycans bear unusual monosaccharide building blocks whose proper construction is critical for bacterial fitness, survival, and colonization in the human host. Despite their appeal as therapeutic targets, bacterial glycans are difficult to study due to the presence of rare bacterial monosaccharides that are linked and modified in atypical manners. Their structural complexity ultimately hampers their analytical characterization. This review highlights recent advances in bacterial chemical glycobiology and focuses on the development of chemical tools to probe, perturb, and image bacterial glycans and their biosynthesis. Current technologies have enabled the study of bacterial glycosylation machinery even in the absence of detailed structural information

Fast bacterial growth reduces antibiotic accumulation and efficacy

This is the final version. Available from eLife Sciences Publications via the DOI in this record. Data availability: All data acquired for this study are presented within the manuscript, the supplementary information and the source data files.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.Engineering and Physical Sciences Research Council (EPSRC)Biotechnology & Biological Sciences Research Council (BBSRC)Medical Research CouncilGordon and Betty Moore FoundationEngineering and Physical Sciences Research CouncilWellcome TrustRoyal SocietyH2020 Marie Skłodowska-Curie Action

Bound To Shock: Protection from Lethal Endotoxemic Shock by a Novel, Nontoxic, Alkylpolyamine Lipopolysaccharide Sequestrant

Lipopolysaccharide (LPS), or endotoxin, a structural component of gram-negative bacterial outer membranes, plays a key role in the pathogenesis of septic shock, a syndrome of severe systemic inflammation which leads to multiple-system organ failure. Despite advances in antimicrobial chemotherapy, sepsis continues to be the commonest cause of death in the critically ill patient. This is attributable to the lack of therapeutic options that aim at limiting the exposure to the toxin and the prevention of subsequent downstream inflammatory processes. Polymyxin B (PMB), a peptide antibiotic, is a prototype small molecule that binds and neutralizes LPS toxicity. However, the antibiotic is too toxic for systemic use as an LPS sequestrant. Based on a nuclear magnetic resonance-derived model of polymyxin B-LPS complex, we had earlier identified the pharmacophore necessary for optimal recognition and neutralization of the toxin. Iterative cycles of pharmacophore-based ligand design and evaluation have yielded a synthetically easily accessible N1,mono-alkyl-mono-homologated spermine derivative, DS-96. We have found that DS-96 binds LPS and neutralizes its toxicity with a potency indistinguishable from that of PMB in a wide range of in vitro assays, affords complete protection in a murine model of LPS-induced lethality, and is apparently nontoxic in vertebrate animal models.This work was supported by NIH grant 1R01 AI50107

Examination of the antibacterial activities of some semi-synthetic chalcone-derivatives alone and in combination with polymyxin B.

Bacterial resistance is an increasing challenge across the world, which means that there is an urgent need for the rational development of antibacterial compounds that have either novel or multiple mechanisms of action. This study demonstrated that two chalcone derivatives - F1 and F23 - had MICs from 16 mg/ml to over 512 mg/ml, when used against two plant pathogens (P. caratovoram and C. michiganensis, subsp. michiganensis) and other important clinical bateria. Both compounds also demonstrated an MIC of 32 mg/ml when used against the quinolone-resistant S. aureus. Though individually weak in terms of their activity, each semi-synthetic agent also displayed notable synergistic action when used in conjunction with polymyxin B against S. aureus, C. violaceum, E. coli and PS. aeruginosa. In these cases, they demonstrated FICs from less than 0.093 to 2. The compounds also demonstrated a FIC index of less than 0.093 when applied with polymyxin B against Neisseriaceae C. violaceum, and were found to have the capacity to extend the spectrum of activity of polymyxin B to include Gram-positive S. aureus species. F1 was found to inhibit the replication of staphylococcal in broth. Combining either F1 or F23 with polymyxin B was found to institute a metabolic blockage in S. aureus and other bacterial species, as determined through a modified MTT reduction assay. The combined agents inflicted major disruptions to the S. aureus cytoplasmic membrane bilayer, as evidenced by the release of intracellular potassium and the influx of Sytox Green fluorescent stain. Notable levels of potential dissipation in the cell membrane, leakage of intracellular potassium ions and blockage of reducing enzymes' activities all occurred within the first thirty minutes. This was far sooner than any significant loss in cell viability - usually recorded after four to eight hours - suggesting that these activities were prerequisites to cell death. In erythrocyte lysis assay, the lowest degree of haemolysis was displayed by the synergistic combinations of 128 mg/ml of either F1 or F23 with 128 mg/ml of polymyxin B, followed by that occurring with 32 mg/ml of either compound combined with 256 mg/ml of the polypeptide antibiotic. In conclusion, further modifications aimed at improving the aqueous solubility of these chalcone derivatives - as well as the antibacterial activity recorded for certain combination concentrations of polymyxin B with either of these semi-synthetic agents - may be required before potential external formulations can be considered. Such preparations may include antiseptic creams, lotions, ointments or aerosols that could be applied with nebulizers in targeted delivery for cases such as cystic fibrosis