Accumulation of Azole Drugs in the Fungal Plant Pathogen Magnaporthe oryzae Is the Result of Facilitated Diffusion Influx

Abstract

Magnaporthe oryzae is an agricultural mold that causes disease in rice, resulting in devastating crop losses. Since rice is a world-wide staple food crop, infection by M. oryzae poses a serious global food security threat. Fungicides, including azole antifungals, are used to prevent and combat M. oryzae plant infections. The target of azoles is CYP51, an enzyme localized on the endoplasmic reticulum (ER) and required for fungal ergosterol biosynthesis. However, many basic drug-pathogen interactions, such as how the azole gets past the fungal cell wall and plasma membrane, and is transported to the ER, are not understood. In addition, reduced intracellular accumulation of antifungals has consistently been observed as a drug resistance mechanism in many fungal species. Studying the basic biology of drug-pathogen interactions may elucidate uncharacterized mechanisms of drug resistance and susceptibility in M. oryzae and potentially other related fungal pathogens. We characterized intracellular accumulation of azole drugs in M. oryzae using a radioactively labeled fluconazole uptake assay to gain insight on whether azoles enter the cell by passive diffusion, active transport, or facilitated diffusion. We show that azole accumulation is not ATP-dependent, nor does it rely on a pH-dependent process. Instead there is evidence for azole drug uptake in M. oryzae by a facilitated diffusion mechanism. The uptake system is specific for azole or azole-like compounds and can be modulated depending on cell phase and growth media. In addition, we found that co-treatment of M. oryzae with ‘repurposed’ clorgyline and radio-labeled fluconazole prevented energy-dependent efflux of fluconazole, resulting in an increased intracellular concentration of fluconazole in the fungal cell.

Synthetic Pesticides Used in Agricultural Production Promote Genetic Instability and Metabolic Variability in Candida spp.

“…Slight accumulations in the cell biomass of C. tropicalis and C. albicans were observed in the case of epoxiconazole and of fenpropimorph in C. glabrata . In our study, the decrease in amounts of Tango ® active compounds observed in Candida cells confirms the observations of Esquivel et al [ 45 ] indicating that reduced intracellular accumulation of antifungal agents is a mechanism of drug resistance in many species of fungi. Intracellular accumulation of azole drugs in Magnaporthe oryzae depended on the nutrient composition as well as the cell phase of the culture.…”

“…Adaptation of the culture to various growth media can modulate the composition of the cytoplasmic membrane in cells, and consequently affect azole uptake. In another study, glucose-containing media reduced final drug accumulation levels, probably due to the activation of glucose-dependent efflux pumps [ 45 ], which would confirm our results for the active substances in Tango ® . Moreover, in studies conducted by Mansfield et al [ 46 ], fluconazole accumulation was inversely correlated with the expression of ATP-dependent efflux pumps in energized C. albicans cells.…”

“…Antagonistic effects have been found when compounds are mixed. Esquivel et al [ 45 ] indicated that antagonism between active compounds may be the result of competition for the import of active compounds into host cells. In the case of insecticides, reduced cytotoxicity is observed in all tested species, when treated either with commercial preparations and/or with the active compounds included in them.…”

Characterization of the Efflux Capability and Substrate Specificity of Aspergillus fumigatus PDR5-like ABC Transporters Expressed in Saccharomyces cerevisiae

“…It reverses FLC resistance in S. cerevisiae strains expressing C. albicans and C. glabrata ABC transporters (31). Clorgyline also inhibits energy-dependent efflux of [ 3 H]FLC in M. oryzae (30). These results indicate that the mechanism of transport inhibition by clorgyline is broadly conserved throughout yeast and filamentous fungi.…”

“…Previous analysis revealed measurable, energy-dependent efflux of fluconazole in A. fumigatus and the plant pathogen Magnaporthe oryzae (29,30). Filamentous fungi contain a higher number of genes encoding predicted ABC transporters than do yeast species such as Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Candida glabrata, and Candida krusei (13,14,17,18).…”