Microbiology (2002), 148, 2617–2625 Printed in Great Britain Molecular and genetic analysis of the Cryptococcus neoformans MET3 gene and a met3 mutant Zhonghui Yang, 1 Renata C. Pascon, 1 J. Andrew Alspaugh, 2 Gary M. Cox 2 and John H. McCusker 1,3 Author for correspondence : John H. McCusker. Tel : ›1 919 681 6744. Fax: ›1 919 684 8735. e-mail : mccus001!mc.duke.edu Departments of Microbiology 1 , Medicine 2 and Genetics 3 , Duke University Medical Center, Durham, NC 27710, USA 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 M . 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 INTRODUCTION The methionine}cysteine biosynthetic pathway is highly complex. This complexity, and the important roles of the sulfur-containing amino acids methionine and cys- teine in the cell, have resulted in the methionine}cysteine pathway being the focus of a major amount of work in saprophytic fungi (reviewed by Jones & Fink, 1982 ; Marzluf, 1997 ; Thomas & Surdin-Kerjan, 1997). The methionine}cysteine biosynthetic pathway is also of considerable interest in the pathogenic fungi. For example, the saprobic mycelial form of the human pathogen Histoplasma capsulatum is prototrophic while the pathogenic yeast form requires cysteine (reviewed by Boguslawski & Stetler, 1979 ; Maresca & Kobayashi, 1989). Similar results are seen in two other dimorphic pathogenic fungi infecting humans, Blastomyces derma- ................................................................................................................................................. Abbreviation : 5FOA, 5-fluoroorotic acid. The GenBank accession numbers for the sequences reported in this paper are AY035556 and AF489498. titidis and Paracoccidioides brasiliensis (Medoff et al., 1987). In addition, the methionine}cysteine biosynthetic pathway is of interest from the perspective of antifungal drug development. Indeed, the antifungal agent azoxy- bacilin inhibits growth by repressing transcription of fungal sulfate-assimilation enzymes of the methionine} cysteine biosynthetic pathway (Aoki et al., 1996). For our initial focus on the methionine}cysteine biosyn- thetic pathway in the human-pathogenic fungus Crypto- coccus neoformans, we decided to examine MET3, which encodes ATP sulfurylase, the first step in the sulfate-assimilation arm of the pathway. One reason for this initial focus on Met3p was a previous report describing hypersensitivity to heat shock of a Sacch- aromyces cerevisiae met3 mutant (Jakubowski & Goldman, 1993). We report the cloning of the C. neoformans MET3 cDNA by complementation of the corresponding S. cerevisiae met3 mutant as well as the construction and reconstitution of a C. neoformans met3 mutant. We also report the phenotypic analysis of the C. neoformans met3 mutant ; specifically, we have determined the effect of the met3 mutation on capsule 0002-5482 # 2002 SGM 2617