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Updated: Jun 26, 2025
Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes
Published on: March 2, 2020
Exploring the Localization of Siderophore-Mediated Cargo Delivery in Gram-Negative Bacteria Using
Yong-Jun Huang1, Ming-Han Yang1, Ling-Yin Lin1
1International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chinese Ministry of Education (MOE), College of Pharmacy, Jinan University, #855 Xingye Avenue, Guangzhou 511400, China.
Abstract:
The design of siderophore-antibiotic conjugates is a promising strategy to overcome drug resistance in negative bacteria. However, accumulating studies have shown that only those antibiotics acting on the cell wall or cell membrane multiply their antibacterial effects when coupled with siderophores, while antibiotics acting on targets in the cytoplasm of bacteria do not show an obvious enhancement of their antibacterial effects when coupled with siderophores. To explore the causes of this phenomenon, we synthesized several conjugate probes using 3-hydroxypyridin-4(1H)-ones as siderophores and replacing the antibiotic cargo with 5-carboxyfluorescein (5-FAM) or malachite green (MG) cargo. By monitoring changes in the fluorescence intensity of FAM conjugate 20 in bacteria, the translocation of the conjugate across the outer membranes of Gram-negative pathogens was confirmed. Further, the use of the fluorogen activating protein(FAP)/MG system revealed that 3-hydroxypyridin-4(1H)-one-MG conjugate 26 was ultimately distributed mainly in the periplasm rather than being translocated into the cytosol of Escherichia coli and Pseudomonas aeruginosa PAO1. Additional mechanistic studies suggested that the uptake of the conjugate involved the siderophore-dependent iron transport pathway and the 3-hydroxypyridin-4(1H)-ones siderophore receptor-dependent mechanism. Meanwhile, we demonstrated that the conjugation of 3-hydroxypyridin-4(1H)-ones to the fluorescein 5-FAM can reduce the possibility of the conjugates crossing the membrane layers of mammalian Vero cells by passive diffusion, and the advantages of the mono-3-hydroxypyridin-4(1H)-ones as a delivery vehicle in the design of conjugates compared to the tri-3-hydroxypyridin-4(1H)-ones. Overall, this work reveals the localization rules of 3-hydroxypyridin-4(1H)-ones as siderophores to deliver the cargo into Gram-negative bacteria. It provides a theoretical basis for the subsequent design of siderophore-antibiotic conjugates, especially based on 3-hydroxypyridin-4(1H)-ones as siderophores.
Insights
This study reveals how 3-hydroxypyridin-4(1H)-ones deliver cargo into Gram-negative bacteria, primarily to the periplasm. This finding is crucial for designing effective siderophore-antibiotic conjugates to combat bacterial resistance.
Area of Science:
- Microbiology
- Drug Discovery
- Bioconjugation Chemistry
Background:
- Siderophore-antibiotic conjugates show promise for overcoming bacterial drug resistance.
- Antibiotic efficacy enhancement depends on the antibiotic's cellular target (cell wall/membrane vs. cytoplasm).
- Understanding the localization of siderophore conjugates is key to their design.
Purpose of the Study:
- To investigate the localization of 3-hydroxypyridin-4(1H)-one conjugates in Gram-negative bacteria.
- To elucidate the mechanism of uptake and translocation of these conjugates.
- To provide a theoretical basis for designing improved siderophore-antibiotic conjugates.
Main Methods:
- Synthesis of 3-hydroxypyridin-4(1H)-one conjugates with fluorescent dyes (5-FAM, malachite green).
- Monitoring fluorescence to track conjugate translocation across bacterial membranes.
- Utilizing the fluorogen activating protein/malachite green system for localization studies.
- Investigating uptake mechanisms via iron transport pathways and specific receptors.
Main Results:
- Conjugate translocation across the outer membrane of Gram-negative pathogens was confirmed.
- 3-hydroxypyridin-4(1H)-one-malachite green conjugates localized primarily in the periplasm, not the cytosol.
- Conjugation reduced passive diffusion into mammalian cells.
- Mono-substituted siderophores showed advantages over tri-substituted ones for delivery.
Conclusions:
- 3-hydroxypyridin-4(1H)-ones deliver cargo to the periplasm of Gram-negative bacteria via specific uptake pathways.
- The localization rules are established for 3-hydroxypyridin-4(1H)-one-based conjugates.
- This research provides a foundation for designing effective siderophore-antibiotic conjugates targeting Gram-negative bacteria.
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