Author: Brooke D. Esquivel

Publisher:

Published: 2016

Total Pages: 0

ISBN-13:

Pathogenic fungi cause serious disease and even death in humans, animals and plants. In medicine and agriculture alike, fungal infections are widespread and represent a significant threat to global public health. The number and array of fungal species, each exhibiting diverse mechanisms of pathogenesis, makes the challenge of fungal infection prevention and treatment formidable. The current repertoire of effective antifungal treatment strategies is very limited. As a result of increased use of antifungals to treat and prevent clinical fungal infections in humans, as well as widespread use of fungicides in agriculture, fungal strains that are resistant to each of the classes of antifungals have emerged. A significant rise in the number of fungal infections in recent years, combined with an increasing amount of drug resistant fungal strains is great cause for concern and places urgency on the development of new and more effective fungal infection treatment and prevention strategies. New fungal drug targets may be discovered with a better understanding of basic fungal biological processes. New or improved fungal infection treatment strategies may stem from a more complete knowledge of fungal response to drug treatment, worldwide trends of fungal pathogenesis and development of resistance, and even fungal evolutionary relationships. The goal of this research was to characterize the most basic fungal/drug interactions, which includes the balance of uptake, retention, and efflux of antifungal drugs in the fungal cell. We analyzed a variety of environmental and cellular factors that affect antifungal drug uptake and retention in two medically and agriculturally important pathogenic fungi, Aspergillus fumigatus and Magnaporthe oryzae. We then identified and characterized a number of A. fumigatus plasma membrane ABC transporters that may contribute to antifungal drug resistance due to their role in the efflux of antifungal drugs. To analyze antifungal drug uptake and retention, we developed an assay to directly measure accumulation of radioactively-labeled azoles in A. fumigatus and M. oryzae. Our analysis of drug uptake under a variety of cellular and environmental conditions demonstrated that these filamentous fungi import azoles by a facilitated diffusion mechanism. Contrasts between the M. oryzae and A. fumigatus data revealed interesting differences that suggest variations in expression, induction, or function of efflux transporters in the two organisms. To analyze antifungal efflux, we cloned and expressed a selection of putative ABC transporter genes from the A. fumigatus genome and heterologously expressed each gene in S. cerevisiae for direct characterization of drug efflux potential. Our efflux transporter analysis showed differences in substrate specificity, drug susceptibilities, energy-dependent efflux activity, and effect of efflux-inhibitor treatment between the different transporters. These data illustrate the complexity of predicting and counteracting fungal drug treatment response, but also highlight the possibilities for identifying new drug targets.