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Molecular and genetic analysis of the Cryptococcus neoformans MET3 gene and a met3 mutant^a

Departments of Microbiology¹, Medicine² and Genetics³, Duke University Medical Center, Durham, NC 27710, USA

Author for correspondence: John H. McCusker. Tel: +1 919 681 6744. Fax: +1 919 684 8735. e-mail: mccus001{at}mc.duke.edu

The Cryptococcus neoformans MET3 cDNA (encoding ATP sulfurylase) was cloned by complementation of the corresponding met3 mutation in Saccharomyces cerevisiae. Sequence analysis showed high similarity between the deduced amino acid sequence of the C. neoformans Met3p and other fungal ATP sulfurylases. A C. neoformans met3 mutant was made by targeted insertional mutagenesis, which had the expected auxotrophic phenotype, and reconstituted the met3 mutant to Met⁺. In vitro, the C. neoformans met3 mutant had a substantial defect in melanin formation, significantly reduced growth rate, and greatly increased thermotolerance. In the murine inhalation infection model, the met3 mutant was avirulent and was deficient in its ability to survive in mice. It is concluded that, in contrast to the yeast form of Histoplasma capsulatum, in C. neoformans the sulfate-assimilation arm of the methionine biosynthetic pathway plays an important role in vitro, even in the presence of abundant exogenous methionine, and is critical for virulence, and indeed for survival, in vivo.

Keywords: Cryptococcus neoformans, methionine auxotroph, ATP sulfurylase, virulence

Abbreviations: 5FOA, 5-fluoroorotic acid

^a The GenBank accession numbers for the sequences reported in this paper are AY035556 and AF489498.

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