Loading...
Subcellular Quantification of Uptake in Gram-Negative Bacteria.
Prochnow, Hans Fetz, Verena Hotop, Sven-Kevin García-Rivera, Mariel A Heumann, Axel
Prochnow, Hans
Fetz, Verena
Hotop, Sven-Kevin
García-Rivera, Mariel A
Heumann, Axel
Citations
Altmetric:
Advisors
Editors
Other Contributors
Issue Date
2019-02-05
Submitted date
Files
Loading...
original manuscript
Adobe PDF, 1.6 MB
Loading...
supporting information
Adobe PDF, 2.37 MB
Other Titles
Abstract
Infections by Gram-negative pathogens represent a major health care issue of growing concern due to a striking lack of novel antibacterial agents over the course of the last decades. The main scientific problem behind the rational optimization of novel antibiotics is our limited understanding of small molecule translocation into, and their export from, the target compartments of Gram-negative species. To address this issue, a versatile, label-free assay to determine the intracellular localization and concentration of a given compound has been developed for Escherichia coli and its efflux-impaired ΔTolC mutant. The assay applies a fractionation procedure to antibiotic-treated bacterial cells to obtain periplasm, cytoplasm, and membrane fractions of high purity, as demonstrated by Western Blots of compartment-specific marker proteins. This is followed by an LC-MS/MS-based quantification of antibiotic content in each compartment. Antibiotic amounts could be converted to antibiotic concentrations by assuming that an E. coli cell is a cylinder flanked by two half spheres and calculating the volumes of bacterial compartments. The quantification of antibiotics from different classes, namely ciprofloxacin, tetracycline, trimethoprim, and erythromycin, demonstrated pronounced differences in uptake quantities and distribution patterns across the compartments. For example, in the case of ciprofloxacin, a higher amount of compound was located in the cytoplasm than in the periplasm (592 ± 50 pg vs 277 ± 13 pg per 3.9 × 10
Citation
Anal Chem. 2019 Feb 5;91(3):1863-1872. doi: 10.1021/acs.analchem.8b03586. Epub 2018 Nov 11
Publisher
Journal
PubMed ID
PubMed Central ID
Additional Links
Embedded video
Type
Article
Language
en
Description
Series/Report no.
ISSN
1520-6882
EISSN
ISBN
ISMN
Gov't Doc #
Sponsors
License
Attribution-NonCommercial-ShareAlike 4.0 International