Exploring a Link between Calcineurin and General Amino Acid Permeases in Candida albicans as a Potential Panfungal Therapeutic Avenue
Human fungal pathogens constitute a large number of species mainly distributed across three phyla, namely Ascomycetes, Basidiomycetes, and Zygomycetes. Many fungi are either inherently resistant or can develop resistance to the current limited number of antifungal drugs. To circumvent this problem, many studies have looked at alternate modes of treatment. One such avenue is inhibiting the calcineurin pathway and thereby the various cellular processes it regulates. Prior work revealed that when the immunosuppressant FK506, a calcineurin inhibitor, was administered to the fungal pathogen Mucor circinelloides, mutants will develop that bypass the inhibition to resume growth in the filamentous form. Characterization of these bypass mutants revealed that upregulation of a general amino acid permease (GAP) plays a key role in FK506 sensitivity. Preliminary data suggest that this phenomenon may also be true in Cryptococcus neoformans (a basidiomycete). The purpose of this project was to determine whether a similar circuit exists in the ascomycete fungal pathogen Candida albicans (because identifying a novel resistance mechanism conserved across all three major phyla would provide an opportunity to develop a pan-fungal therapeutic option). To that end, homozygous gap2Δ/Δ mutants were constructed in the C. albicans wild-type strain SC5314 utilizing the SAT-FLP cassette. The mutant yeast strains were verified both genotypically (via Southern blot analysis) and phenotypically (by assaying growth on various nitrogenous sources). Next, the potential role of Gap2 in calcineurin function was evaluated by assaying the ability of the various strains to grow in serum with and without FK506. Unfortunately, the results obtained suggest that there may be compensatory activity from other GAP genes under some conditions. Future endeavors should include an analysis of C. albicans mutants lacking each of the GAP genes and performing quantitative RT-PCR on these to determine whether it might be necessary to delete more than one gene in order to see the same FK506 resistance phenotype identified in the other fungal species.