title: De NovoPyrimidine Biosynthesis Connects Cell Integrity to Amphotericin B Susceptibility inCryptococcus neoformans.
abstract: 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 aCryptococcus 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-typeURA1gene 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 theura1Δ mutant. We likewise saw an increased rate of DiOC6association in other AmB-sensitive mutants, including aura-spontaneousURA5mutant made by 5-fluoroorotic acid (5-FOA) selection and abck1Δ mutant defective in cell integrity signaling. Similar results were also obtained by using a specific plasma membrane-binding CellMask live stain, with cell integrity mutants that exhibited increased and faster association of the dye with the membrane. Chitin synthase mutants (chs5Δ andchs6Δ) 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 AmBin vitro.IMPORTANCESynergy between AmB and nucleotide biosynthetic pathways has been documented, but the mechanism of this interaction has not been delineated. Results from this study suggest a correlation between uridine nucleotide biosynthesis and cell integrity likely mediated through the pool of nucleotide-sugar conjugates, which are precursor molecules for both capsule and cell wall ofC. neoformans. Thus, we propose a mechanism by which structural defects in the cell wall resulting from perturbation of pyrimidine biosynthesis allow faster and increased penetration of AmB molecules into the cell membrane. Overall, our work demonstrates that impairment of pyrimidine biosynthesis inC. neoformanscould be a potential target for antifungal therapy, either alone or in combination with AmB.
authors.affiliation: Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, The State University of New York, Buffalo, New York, USA.
authors.forename: Dithi
authors.initials: D
authors.lastname: Banerjee
authors.affiliation: Department of Structural Biology, Hauptman Woodward Medical Research Institute, University at Buffalo, The State University of New York, Buffalo, New York, USA.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Umland
authors.affiliation: Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, The State University of New York, Buffalo, New York, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Panepinto
keywords: amphotericin B; cell wall integrity; pyrimidine biosynthesis