Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

An azido-oxazolidinone antibiotic for live bacterial cell imaging and generation of antibiotic variants.

ABSTRACT: An azide-functionalised analogue of the oxazolidinone antibiotic linezolid was synthesised and shown to retain antimicrobial activity. Using facile 'click' chemistry, this versatile intermediate can be further functionalised to explore antimicrobial structure-activity relationships or conjugated to fluorophores to generate fluorescent probes. Such probes can report bacteria and their location in a sample in real time. Modelling of the structures bound to the cognate 50S ribosome target demonstrates binding to the same site as linezolid is possible. The fluorescent probes were successfully used to image Gram-positive bacteria using confocal microscopy.

SUBMITTER: Phetsang W

PROVIDER: S-EPMC4141890 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Second-generation covalent TMP-tag for live cell imaging.

Project description:Chemical tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with organic fluorophores that have improved brightness and other specialized properties. Recently, we successfully rendered our TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow in vitro labeling kinetics and limited live cell protein labeling. Thus, here we report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical synthesis for routine production of a variety of A-TMP-probe v2.0 labels. We then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 in vitro. Finally, we demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited number of chemical tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and organic chemistry as tools not only for chemical tag engineering but also more broadly for synthetic biology.

| S-EPMC3433398 | biostudies-literature

Click-free imaging of carbohydrate trafficking in live cells using an azido photothermal probe.

Project description:Real-time tracking of intracellular carbohydrates remains challenging. While click chemistry allows bio-orthogonal tagging with fluorescent probes, the reaction permanently alters the target molecule and only allows a single snapshot. Here, we demonstrate click-free mid-infrared photothermal (MIP) imaging of azide-tagged carbohydrates in live cells. Leveraging the micromolar detection sensitivity for 6-azido-trehalose (TreAz) and the 300-nm spatial resolution of MIP imaging, the trehalose recycling pathway in single mycobacteria, from cytoplasmic uptake to membrane localization, is directly visualized. A peak shift of azide in MIP spectrum further uncovers interactions between TreAz and intracellular protein. MIP mapping of unreacted azide after click reaction reveals click chemistry heterogeneity within a bacterium. Broader applications of azido photothermal probes to visualize the initial steps of the Leloir pathway in yeasts and the newly synthesized glycans in mammalian cells are demonstrated.

| S-EPMC10979903 | biostudies-literature

Tedizolid Phosphate: a Next-Generation Oxazolidinone.

Project description:Treatment of multidrug-resistant Gram-positive infections continues to challenge clinicians as the emergence of new resistance mechanisms outpaces introduction of novel antimicrobial agents. Tedizolid phosphate is a next-generation oxazolidinone with activity against both methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus spp. Tedizolid has consistently shown potency advantages over linezolid against Gram-positive microorganisms including those with reduced susceptibility to linezolid. Of particular significance, minimum inhibitory concentrations of tedizolid appear to be largely unaffected by the chloramphenicol-florfenicol resistance (cfr) gene, which has been implicated in a number of published linezolid-resistant organism outbreaks. Tedizolid phosphate also has been found to have a favorable pharmacokinetic profile allowing for once-daily dosing in both oral and intravenous forms. Potency and pharmacokinetic advantages have allowed for lower total daily doses of tedizolid, compared to linezolid, being needed for clinical efficacy in the treatment of acute bacterial skin and skin structure infections (ABSSSI). The decreased total drug exposure produced may in part be responsible for a decrease in the observed adverse effects including thrombocytopenia. Tedizolid phosphate is currently indicated for the treatment of ABSSSI and under investigation for the treatment of nosocomial pneumonia. Although much of the role of tedizolid remains to be defined by expanding clinical experience, tedizolid is likely a welcomed addition to the mere handful of agents available for the treatment of multidrug-resistant Gram-positive infections.

| S-EPMC4363212 | biostudies-literature

Tedizolid phosphate (sivextro): a second-generation oxazolidinone to treat acute bacterial skin and skin structure infections.

Project description:Tedizolid phosphate: a second-generation oxazolidinone for acute bacterial skin and skin structure infections.

| S-EPMC4123804 | biostudies-other

Single-molecule imaging reveals modulation of cell wall synthesis dynamics in live bacterial cells.

Project description:The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability. Proper cell wall construction requires the interaction of synthesis enzymes and the cytoskeleton, but it is unclear how the activities of individual proteins are coordinated to preserve the morphology and integrity of the cell wall during growth. To elucidate this coordination, we used single-molecule imaging to follow the behaviours of the two major peptidoglycan synthases in live, elongating Escherichia coli cells and after perturbation. We observed heterogeneous localization dynamics of penicillin-binding protein (PBP) 1A, the synthase predominantly associated with cell wall elongation, with individual PBP1A molecules distributed between mobile and immobile populations. Perturbations to PBP1A activity, either directly through antibiotics or indirectly through PBP1A's interaction with its lipoprotein activator or other synthases, shifted the fraction of mobile molecules. Our results suggest that multiple levels of regulation control the activity of enzymes to coordinate peptidoglycan synthesis.

| S-EPMC5078992 | biostudies-literature

A live-cell super-resolution technique demonstrated by imaging germinosomes in wild-type bacterial spores.

Project description:Time-lapse fluorescence imaging of live cells at super-resolution remains a challenge, especially when the photon budget is limited. Current super-resolution techniques require either the use of special exogenous probes, high illumination doses or multiple image acquisitions with post-processing or combinations of the aforementioned. Here, we describe a new approach by combining annular illumination with rescan confocal microscopy. This optics-only technique generates images in a single scan, thereby avoiding any potential risks of reconstruction related artifacts. The lateral resolution is comparable to that of linear structured illumination microscopy and the axial resolution is similar to that of a standard confocal microscope. As a case study, we present super-resolution time-lapse imaging of wild-type Bacillus subtilis spores, which contain low numbers of germination receptor proteins in a focus (a germinosome) surrounded by an autofluorescent coat layer. Here, we give the first evidence for the existence of germinosomes in wild-type spores, show their spatio-temporal dynamics upon germinant addition and visualize spores coming to life.

| S-EPMC7093444 | biostudies-literature

Quantitative live-cell imaging of secretion activity reveals dynamic immune responses

Project description:Quantification of cytokine secretion has facilitated advances in the field of immunology, yet the dynamic and varied secretion profiles of individual cells, particularly those obtained from limited human samples, remain obscure. Herein, we introduce a new technology for quantitative live-cell imaging of secretion activity (qLCI-S) that enables high-throughput and dual-color monitoring of secretion activity at the single-cell level over several days, followed by transcriptome analysis of individual cells based on their phenotype. The efficacy of qLCI-S was demonstrated by visualizing the characteristic temporal pattern of cytokine secretion of group 2 innate lymphoid cells, which constitute less than 0.01% of human peripheral blood mononuclear cells, and by revealing minor subpopulations with enhanced cytokine production. The underlying mechanism of this feature was linked to the gene expression of stimuli receptors. This new technology paves the way for exploring gene expression signatures linked to the spatiotemporal dynamic nature of various secretory functions.

2024-02-01 | GSE253100 | GEO

A Protocol to Engineer Bacteriophages for Live-Cell Imaging of Bacterial Prophage Induction Inside Mammalian Cells.

Project description:The gut microbiome is dominated by lysogens, bacteria that carry bacterial viruses (phages). Uncovering the function of phages in the microbiome and observing interactions between phages, bacteria, and mammalian cells in real time in specific cell types are limited by the difficulty of engineering fluorescent markers into large, lysogenic phage genomes. Here, we present a method to multiplex the engineering of life-cycle reporters into lysogenic phages and how to infect macrophages with engineered lysogens to study these interactions at the single-cell level. For complete details on the use and execution of this protocol, please refer to Bodner et al. (2020).

| S-EPMC7580223 | biostudies-literature

Live cell imaging 72 hour screening

Project description: Not available

| S-TOXR1741 | biostudies-other

Live cell imaging 72 hour screening

Project description: Not available

| S-TOXR1743 | biostudies-other