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Eukaryotic Cell, March 2009, p. 315-326, Vol. 8, No. 3
1535-9778/09/$08.00+0     doi:10.1128/EC.00375-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of ENA1 as a Virulence Gene of the Human Pathogenic Fungus Cryptococcus neoformans through Signature-Tagged Insertional Mutagenesis {triangledown}

Alexander Idnurm,1,2* Felicia J. Walton,1,{dagger} Anna Floyd,1 Jennifer L. Reedy,1 and Joseph Heitman1*

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710,1 Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri—Kansas City, Kansas City, Missouri 641102

Received 26 November 2008/ Accepted 8 January 2009

A library of more than 4,500 signature-tagged insertion mutants of the human pathogenic fungus Cryptococcus neoformans was generated, and a subset was screened in a murine inhalation model to identify genes required for virulence. New genes that regulate aspects of C. neoformans virulence were also identified by screening the entire library for in vitro phenotypes related to the ability to cause disease, including melanin production, growth at high temperature, and growth under conditions of nutrient limitation. A screen of 10% of the strain collection in mice identified an avirulent mutant strain with an insertion in the ENA1 gene, which is predicted to encode a fungus-specific sodium or potassium P-type ATPase. The results of the deletion of the gene and complementation experiments confirmed its key role in mammalian virulence. ena1 mutant strains exhibited no change in sensitivity to high salt concentrations but were sensitive to alkaline pH conditions, providing evidence that the fungus may have to survive at elevated pH during infection of the mammalian host. The mutation of the well-characterized virulence factor calcineurin (CNA1) also rendered C. neoformans strains sensitive to elevated pH. ENA1 transcripts in wild-type and cna1 mutant strains were upregulated in response to high pH, and cna1 ena1 double mutant strains exhibited increased sensitivity to elevated pH, indicating that at least two pathways in the fungus mediate survival under alkaline conditions. Signature-tagged mutagenesis is an effective strategy for the discovery of new virulence genes in fungal pathogens of animals.

* Corresponding author. Mailing address for Alexander Idnurm: Room 513, Spencer Chemistry Building, 5100 Rockhill Rd., School of Biological Sciences, Division of Cell Biology and Biophysics, University of Missouri—Kansas City, Kansas City, MO 64110. Phone: (816) 235-2265. Fax: (816) 235-1503. E-mail: idnurma{at}umkc.edu. Mailing address for Joseph Heitman: Room 322, CARL Building, Box 3546, Research Dr., Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-2428. Fax: (919) 684-5458. E-mail: heitm001{at}duke.edu

{triangledown} Published ahead of print on 16 January 2009.

{dagger} Present address: The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, United Kingdom.

Eukaryotic Cell, March 2009, p. 315-326, Vol. 8, No. 3
1535-9778/09/$08.00+0     doi:10.1128/EC.00375-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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