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Published on: November 28, 2019
Transport Across Membranes: Techniques for Measuring Drug Import in Fungal Cells
Brooke D Esquivel1, Theodore C White2
1Department of Cell and Molecular Biology and Biochemistry, School of Biological and Chemical Sciences, University of Missouri at Kansas City, Kansas City, MO, USA. bdp3k9@mail.umkc.edu.
Abstract:
The ability of many antifungal molecules to traverse the fungal cell wall and accumulate within the cell is crucial to its ability to have the desired biological activity. Altered accumulation of the drug is an important mechanism of antifungal drug resistance. The best characterized mechanism for altered accumulation is through the action of the drug efflux pump which actively transports the drugs out of the membrane, although this is not the only mechanism for this phenomenon. Here, we describe protocols for the measurement of uptake of tritiated fluconazole in both yeast and filamentous fungi.
Insights
Measuring antifungal drug uptake is key to understanding drug resistance. This study details methods for quantifying fluconazole accumulation in fungi, aiding research into drug efficacy and resistance mechanisms.
Area of Science:
- Mycology
- Pharmacology
- Biochemistry
Background:
- Antifungal drug efficacy relies on cellular accumulation.
- Altered drug accumulation, particularly via efflux pumps, is a major cause of antifungal drug resistance.
- Understanding drug uptake mechanisms is crucial for developing effective antifungal therapies.
Purpose of the Study:
- To describe standardized protocols for measuring the uptake of antifungal drugs in fungal cells.
- To quantify the intracellular accumulation of tritiated fluconazole in both yeast and filamentous fungal models.
- To provide a foundation for investigating drug resistance mechanisms related to altered drug accumulation.
Main Methods:
- Development and validation of protocols for measuring tritiated fluconazole uptake.
- Application of these protocols to representative yeast and filamentous fungal species.
- Quantification of intracellular drug concentrations using radiolabeling techniques.
Main Results:
- Established reliable methods for assessing fluconazole uptake in diverse fungal forms.
- Demonstrated the feasibility of quantifying drug accumulation in both yeast and filamentous fungi.
- Provided quantitative data on drug uptake essential for resistance studies.
Conclusions:
- The described protocols enable accurate measurement of antifungal drug uptake.
- These methods are vital for studying the role of accumulation in antifungal activity and resistance.
- This work facilitates further research into overcoming antifungal drug resistance.
