Fluorescent aminoglycoside antibiotics and methods for accurately monitoring uptake by bacteria

Mahnaz Sabeti Azad (1, 2) , Maho Okuda , Mélina Cyrenne (1, 2) , Mickael Bourge (1, 3) , Marie-Pierre Heck , Satoko Yoshizawa (1, 2) , Dominique Fourmy (1, 2)

1 I2BC - Institut de Biologie Intégrative de la Cellule
2 RNAStr - Structure et dynamique des ARN
3 CYTO - Cytométrie

Characterizing 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 non-specific 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 non-permeable 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.

Mots clés: Fluorescence; antibiotics; aminoglycosides; uptake; flow cytometry; microscopy

Domaines: Pharmacologie; Bactériologie

Mahnaz Sabeti Azad, Maho Okuda, Mélina Cyrenne, Mickael Bourge, Marie-Pierre Heck, et al.. Fluorescent aminoglycoside antibiotics and methods for accurately monitoring uptake by bacteria. ACS Infectious Diseases, 2020, 6 (5), pp.1008-1017. ⟨10.1021/acsinfecdis.9b00421⟩. ⟨hal-02516160⟩