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Infection and Immunity, April 1999, p. 1812-1820, Vol. 67, No. 4
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

Maurizio del Poeta,1,2 Dena L. Toffaletti,1 Thomas H. Rude,1 Sara D. Sparks,3 Joseph Heitman,1,4,5,6 and John R. Perfect1,*

Departments of Medicine,1 Genetics,5 and Pharmacology and Cancer Biology6 and the Howard Hughes Medical Institute,4 Duke University Medical Center, Durham, North Carolina 27710; Clinical Flow Cytometry Laboratory, University of North Carolina Hospital at Chapel Hill, Chapel Hill, North Carolina 275993; and Institute of Infectious Diseases and Public Health, University of Ancona, 60121 Ancona, Italy2

Received 4 September 1998/Returned for modification 9 October 1998/Accepted 28 December 1998

Synthetic green fluorescent protein (GFP) was used as a reporter to detect differential gene expression in the pathogenic fungus Cryptococcus neoformans. Promoters from the C. neoformans actin, GAL7, or mating-type alpha pheromone (MFalpha 1) genes were fused to GFP, and the resulting reporter genes were used to assess gene expression in serotype A C. neoformans. Yeast cells containing an integrated pACT::GFP construct demonstrated that the actin promoter was expressed during vegetative growth on yeast extract-peptone-dextrose medium. In contrast, yeast cells containing the inducible GAL7::GFP or MFalpha 1::GFP reporter genes expressed significant GFP activity only during growth on galactose medium or V-8 agar, respectively. These findings demonstrated that the GAL7 and MFalpha 1 promoters from a serotype D C. neoformans strain function when introduced into a serotype A strain. Because the MFalpha 1 promoter is induced by nutrient deprivation and the MATalpha locus containing the MFalpha 1 gene has been linked with virulence, yeast cells containing the pMFalpha 1::GFP reporter gene were analyzed for GFP expression in the central nervous system (CNS) of immunosuppressed rabbits. In fact, significant GFP expression from the MFalpha 1::GFP reporter gene was detected after the first week of a CNS infection. These findings suggest that there are temporal, host-specific cues that regulate gene expression during infection and that the MFalpha 1 gene is induced during the proliferative stage of a CNS infection. In conclusion, GFP can be used as an effective and sensitive reporter to monitor specific C. neoformans gene expression in vitro, and GFP reporter constructs can be used as an approach to identify a novel gene(s) or to characterize known genes whose expression is regulated during infection.

* Corresponding author. Mailing address: Department of Medicine, Duke University Medical Center, Division of Infectious Diseases and International Health, P.O. Box 3353, Durham, NC 27710. Phone: (919) 684-2660. Fax: (919) 684-8902. E-mail: perfe001{at}mc.duke.edu.

Infection and Immunity, April 1999, p. 1812-1820, Vol. 67, No. 4
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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