Molecular and genetic analysis of the Cryptococcus neoformans MET3 gene and a met3 mutant.
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.
Yang
Zhonghui
Z
Departments of Microbiology1, Medicine2 and Genetics3, Duke University Medical Center, Durham, NC 27710, USA.
Pascon
Renata C
RC
Departments of Microbiology1, Medicine2 and Genetics3, Duke University Medical Center, Durham, NC 27710, USA.
Alspaugh
Andrew
A
Departments of Microbiology1, Medicine2 and Genetics3, Duke University Medical Center, Durham, NC 27710, USA.
Cox
Gary M
GM
Departments of Microbiology1, Medicine2 and Genetics3, Duke University Medical Center, Durham, NC 27710, USA.
McCusker
John H
JH
Departments of Microbiology1, Medicine2 and Genetics3, Duke University Medical Center, Durham, NC 27710, USA.
D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003453:Cryptococcosis / Q000382:microbiology; D003455:Cryptococcus neoformans / Q000201:enzymology / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D008715:Methionine / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D013430:Sulfate Adenylyltransferase / Q000235:genetics*; D014774:Virulence
D008715:Methionine; D013430:Sulfate Adenylyltransferase