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Authors
Prochnow, HansFetz, Verena
Hotop, Sven-Kevin
García-Rivera, Mariel A
Heumann, Axel
Brönstrup, Mark

Issue Date
2019-02-05
Metadata
Show full item recordAbstract
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 × 10Citation
Anal Chem. 2019 Feb 5;91(3):1863-1872. doi: 10.1021/acs.analchem.8b03586. Epub 2018 Nov 11Affiliation
HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany.Publisher
ACS PublicationsJournal
Analytical chemistryPubMed ID
30485749Type
ArticleLanguage
enISSN
1520-6882ae974a485f413a2113503eed53cd6c53
10.1021/acs.analchem.8b03586
Scopus Count
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