Abstract
Despite the wealth of knowledge regarding the mechanisms of action and the mechanisms of resistance to azole antifungals, very little is known about how the azoles are imported into pathogenic fungal cells. Here the in-vitro accumulation and import of Fluconazole (FLC) was examined in the pathogenic fungus, Candida albicans. In energized cells, FLC accumulation correlates inversely with expression of ATP-dependent efflux pumps. In de-energized cells, all strains accumulate FLC, suggesting that FLC import is not ATP-dependent. The kinetics of import in de-energized cells displays saturation kinetics with a Km of 0.64 uM and Vmax of 0.0056 pmol/min/108 cells, demonstrating that FLC import proceeds via facilitated diffusion through a transporter rather than passive diffusion. Other azoles inhibit FLC import on a mole/mole basis, suggesting that all azoles utilize the same facilitated diffusion mechanism. An analysis of related compounds indicates that competition for azole import depends on an aromatic ring and an imidazole or triazole ring together in one molecule. Import of FLC by facilitated diffusion is observed in other fungi, including Cryptococcus neoformans, Saccharomyces cerevisiae, and Candida krusei, indicating that the mechanism of transport is conserved among fungal species. FLC import was shown to vary among Candida albicans resistant clinical isolates, suggesting that altered facilitated diffusion may be a previously uncharacterized mechanism of resistance to azole drugs.
Highlights
The incidence of invasive fungal disease has increased over 200% in the US in the last 25 years [1], likely the result of a parallel increase in the immunocompromised patient population
A great deal is known about how the azoles interact with their target enzyme within fungal cells and how the azoles are exported from the fungal cell through various efflux pumps
Azole import by facilitated diffusion is shown in four species of fungi, suggesting that it is common for most if not all fungi
Summary
The incidence of invasive fungal disease has increased over 200% in the US in the last 25 years [1], likely the result of a parallel increase in the immunocompromised patient population. Candida species are the most common invasive fungal pathogens, with Candida albicans accounting for more than 50% of all infections [2]. One of the first lines of defense for treating pathogenic fungal infections are the azole drugs, including FLC, the most commonly used azole. The significant increase in invasive fungal infections and the prolonged and repeated treatment of AIDS patients has resulted in a marked increase in the emergence of FLC-resistant C. albicans isolates [6,7,8]. Clinical isolates have not been investigated for altered azole import as a mechanism of resistance
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