id9b00421_si_001.pdf (9.57 MB)
Fluorescent Aminoglycoside Antibiotics and Methods for Accurately Monitoring Uptake by Bacteria
journal contribution
posted on 2020-03-27, 20:04 authored by Mahnaz Sabeti Azad, Maho Okuda, Mélina Cyrenne, Mickael Bourge, Marie-Pierre Heck, Satoko Yoshizawa, Dominique FourmyCharacterizing
how multidrug-resistant bacteria circumvent the action of clinically
used or novel antibiotics requires a detailed understanding of how
the antibiotics interact with and cross bacterial membranes to accumulate
in the cells and exert their action. When monitoring the interactions
of drugs with bacteria, it remains challenging to differentiate functionally
relevant internalized drug levels from nonspecific binding. Fluorescence
is a method of choice for observing dynamics of biomolecules. In order
to facilitate studies involving aminoglycoside antibiotics, we have
generated fluorescently labeled aminoglycoside derivatives with uptake
and bactericidal activities similar, albeit with a moderate loss,
to those of the parent drug. The method combines fluorescence microscopy
with fluorescence-activated cell sorting (FACS) using neomycin coupled
to nonpermeable cyanine dyes. Fluorescence imaging allowed membrane-bound
antibiotic to be distinguished from molecules in the cytoplasm. Patterns
of uptake were assigned to different populations in the FACS analysis.
Our study illustrates how fluorescent derivatives of an aminoglycoside
enable a robust characterization of the three components of uptake:
membrane binding, EDPI, and EDPII. Because EDPI levels are weak compared
to the two other types of accumulation and critical for the action
of these drugs, the three components of uptake must be taken into
account separately when drawing conclusions about aminoglycoside function.
History
Usage metrics
Categories
Keywords
Fluorescent Aminoglycoside Antibioticsmembrane bindingfluorescence-activated cellmultidrug-resistant bacterianonpermeable cyanine dyesdrug levelsnovel antibioticsmethodmembrane-bound antibioticparent drugEDPIIaminoglycoside antibioticsbactericidal activitiesAccurately Monitoring Uptakeuptakeaminoglycoside derivativesFACS analysisEDPI levelsaminoglycoside functioncomponentfluorescence microscopyFluorescence imaging
Licence
Exports
RefWorks
BibTeX
Ref. manager
Endnote
DataCite
NLM
DC