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Fungal infections are often difficult to treat due to the inherent similarities between fungal and animal cells and the resulting host toxicity from many antifungal compounds. Cryptococcus neoformans is an opportunistic fungal pathogen of humans that causes life-threatening disease, primarily in immunocompromised patients. Since antifungal therapy for this microorganism is limited, many investigators have explored novel drug targets aim at virulence factors, such as the ability to grow at mammalian physiological temperature (37°C). To address this issue, we used the Agrobacterium tumefaciens gene delivery system to create a random insertion mutagenesis library that was screened for altered growth at elevated temperatures. Among several mutants unable to grow at 37°C, we explored one bearing an interruption in the URA4 gene. This gene encodes dihydroorotase (DHOase) that is involved in the de novo synthesis of pyrimidine ribonucleotides. Loss of the C. neoformans Ura4 protein, by targeted gene interruption, resulted in an expected uracil/uridine auxotrophy and an unexpected high temperature growth defect. In addition, the ura4 mutant displayed phenotypic defects in other prominent virulence factors (melanin, capsule and phospholipase) and reduced stress response compared to wild type and reconstituted strains. Accordingly, this mutant had a decreased survival rate in macrophages and attenuated virulence in a murine model of cryptococcal infection. Quantitative PCR analysis suggests that this biosynthetic pathway is induced during the transition from 30°C to 37°C, and that transcriptional regulation of de novo and salvage pyrimidine pathway are under the control of the Ura4 protein., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)