Dataset Information

AICAR transformylase/IMP cyclohydrolase (ATIC) is essential for de novo purine biosynthesis and infection by Cryptococcus neoformans.

ABSTRACT: The fungal pathogen Cryptococcus neoformans is a leading cause of meningoencephalitis in the immunocompromised. As current antifungal treatments are toxic to the host, costly, limited in their efficacy, and associated with drug resistance, there is an urgent need to identify vulnerabilities in fungal physiology to accelerate antifungal discovery efforts. Rational drug design was pioneered in de novo purine biosynthesis as the end products of the pathway, ATP and GTP, are essential for replication, transcription, and energy metabolism, and the same rationale applies when considering the pathway as an antifungal target. Here, we describe the identification and characterization of C. neoformans 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/5'-inosine monophosphate cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final two enzymatic steps in the formation of the first purine base inosine monophosphate. We demonstrate that mutants lacking the ATIC-encoding ADE16 gene are adenine and histidine auxotrophs that are unable to establish an infection in a murine model of virulence. In addition, our assays employing recombinantly expressed and purified C. neoformans ATIC enzyme revealed K_m values for its substrates AICAR and 5-formyl-AICAR are 8-fold and 20-fold higher, respectively, than in the human ortholog. Subsequently, we performed crystallographic studies that enabled the determination of the first fungal ATIC protein structure, revealing a key serine-to-tyrosine substitution in the active site, which has the potential to assist the design of fungus-specific inhibitors. Overall, our results validate ATIC as a promising antifungal drug target.

SUBMITTER: Wizrah MSI

PROVIDER: S-EPMC9525906 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Publications

AICAR transformylase/IMP cyclohydrolase (ATIC) is essential for de novo purine biosynthesis and infection by Cryptococcus neoformans.

Wizrah Maha S I MSI Chua Sheena M H SMH Luo Zhenyao Z Manik Mohammad K MK Pan Mengqi M Whyte Jessica M L JML Robertson Avril A B AAB Kappler Ulrike U Kobe Bostjan B Fraser James A JA

The Journal of biological chemistry 20220905 10

The fungal pathogen Cryptococcus neoformans is a leading cause of meningoencephalitis in the immunocompromised. As current antifungal treatments are toxic to the host, costly, limited in their efficacy, and associated with drug resistance, there is an urgent need to identify vulnerabilities in fungal physiology to accelerate antifungal discovery efforts. Rational drug design was pioneered in de novo purine biosynthesis as the end products of the pathway, ATP and GTP, are essential for replicatio ...[more]

PMID: 36063996

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Project description: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 ura1? 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 ura1? mutant. We likewise saw an increased rate of DiOC6 association in other AmB-sensitive mutants, including a ura- spontaneous URA5 mutant made by 5-fluoroorotic acid (5-FOA) selection and a bck1? 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 (chs5? and chs6?) 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 ?-1,6-glucan synthase inhibitor caspofungin was synergistic with AmB in vitro. IMPORTANCE Synergy 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 of C. 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 in C. neoformans could be a potential target for antifungal therapy, either alone or in combination with AmB.

Dataset Information

AICAR transformylase/IMP cyclohydrolase (ATIC) is essential for de novo purine biosynthesis and infection by Cryptococcus neoformans.

Publications

AICAR transformylase/IMP cyclohydrolase (ATIC) is essential for de novo purine biosynthesis and infection by Cryptococcus neoformans.

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