The role of the de novo pyrimidine biosynthetic pathway in Cryptococcus neoformans high temperature growth and virulence

Abstract

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.

De NovoPyrimidine Biosynthesis Connects Cell Integrity to Amphotericin B Susceptibility in Cryptococcus neoformans

“…4A; P < 0.05), whereas 100% survival was observed in larvae infected with the knockout strains at both temperatures. These data demonstrated that in addition to temperature sensitivity, pyrimidine pool depletion also plays a role in virulence, similarly to results obtained with a ura4 Δ mutant (16). No death was observed in any of the mock-injected controls at either 30°C or 37°C until 5 days postinoculation.…”

“…The malaria parasite also expresses a family 2 enzyme, but the malarial DHODH is not sensitive to brequinar (12, 15), indicating the species specificity of this enzyme. Impairment of de novo pyrimidine biosynthesis by perturbing other enzymes in the pathway impairs cell integrity in C. neoformans and prevents capsule biosynthesis in the absence of exogenous uracil (16, 17). …”

F901318 represents a novel class of antifungal drug that inhibits dihydroorotate dehydrogenase

“…S3). In animal models of infection, pyrimidine biosynthesis mutants from several pathogenic fungi are highly attenuated for virulence, including studies on A. fumigatus (14), C. albicans (15), H. capsulatum (16), and C. neoformans (17). In Saccharomyces cerevisiae, a ura3 deletion strain lacking the orotidine-5′-phosphate (OMP) decarboxylase enzyme of pyrimidine biosynthesis was unable to survive in vivo, exhibiting a decrease in the competitive index versus a WT or reconstituted strain observed after just 4 h (27).…”

“…Interestingly, human serum contains low levels of pyrimidines, estimated to be ∼15 μM (13), which are insufficient to reverse the effect of F901318 on A. fumigatus in vivo. Indeed, mutants of A. fumigatus (14), Candida albicans (15), Histoplasma capsulatum (16), and Cryptococcus neoformans (17), which are disrupted in pyrimidine biosynthesis, have exhibited attenuated virulence in animal models of infection, indicating that targeting pyrimidine synthesis is a valid antifungal strategy.…”

Two-spotted cricket as an animal infection model of human pathogenic fungi

“…C. glabrata alters cell surface polysaccharides and shows different cell surface hydrophobicity at 37°C compared to that at a lower temperature (50,51). C. neoformans upregulates nucleotide metabolism and capsule formation at a high temperature, enabling growth at a high temperature (52,53). Because C. neoformans did not exhibit increased killing activity against crickets at 37°C, the physiologic alteration that is present in the two Candida species but is absent in C. neoformans may contribute to the temperature-dependent killing activity against crickets.…”