De novo pyrimidine biosynthesis connects cell integrity to amphotericin B susceptibility in Cryptococcus neoformans

The use of amphotericin B (AmB) in conjunction with 5-fluorocytosine (5-FC) is known to be the optimal therapy for treating cryptococcosis, but the mechanism by which 5-FC synergizes with AmB is unknown. In this study, we generated a Cryptococcus neoformans ura1Δ mutant lacking dihydroorotate dehydrogenase (DHODH), which demonstrated temperature-sensitive growth due to a defect in cell integrity and sensitivity to cell wall-damaging agents. In addition, sensitivity to AmB was greatly increased. Inclusion of uracil or uridine in the medium did not suppress the cell wall or AmB phenotype, whereas complementation with the wild-type URA1 gene complemented the mutant phenotype. As a measure of membrane accessibility, we assayed the rate of association of the lipid-binding dye 3,3=-dihexyloxacarbocyanine iodide (DiOC₆) and saw more rapid association in the ura1Δ mutant. We likewise saw an increased rate of DiOC₆ association in other AmB-sensitive mutants, including a uraβ spontaneous URA5 mutant made by 5-fluoroorotic acid (5-FOA) selection and a bck1Δ mutant defective in cell integrity signaling. Similar results were also obtained by using a specific plasma membranebinding CellMask live stain, with cell integrity mutants that exhibited increased and faster association of the dye with the membrane. Chitin synthase mutants (chs5Δ and chs6Δ) that lack any reported cell wall defects, in turn, demonstrate neither any increased susceptibility to AmB nor a greater accessibility to either of the dyes. Finally, perturbation of the cell wall of the wild type by treatment with the β-1,6- glucan synthase inhibitor caspofungin was synergistic with AmB in vitro.