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Journal of Bacteriology, January 1999, p. 444-453, Vol. 181, No. 2
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

A Glucan Synthase FKS1 Homolog in Cryptococcus neoformans Is Single Copy and Encodes an Essential Function

John R. Thompson,^1,* Cameron M. Douglas,¹ Weili Li,¹ Chong K. Jue,^1, Barnali Pramanik,¹ Xiling Yuan,¹ Thomas H. Rude,² Dena L. Toffaletti,² John R. Perfect,² and Myra Kurtz¹

Infectious Diseases, Merck Research Laboratories, Rahway, New Jersey 07065,¹ and Department of Medicine, Division of Infectious Diseases, Duke University Medical Center, Durham, North Carolina 27710²

Received 21 August 1998/Accepted 13 November 1998

Cryptococcal meningitis is a fungal infection, caused by Cryptococcus neoformans, which is prevalent in immunocompromised patient populations. Treatment failures of this disease are emerging in the clinic, usually associated with long-term treatment with existing antifungal agents. The fungal cell wall is an attractive target for drug therapy because the syntheses of cell wall glucan and chitin are processes that are absent in mammalian cells. Echinocandins comprise a class of lipopeptide compounds known to inhibit 1,3--glucan synthesis, and at least two compounds belonging to this class are currently in clinical trials as therapy for life-threatening fungal infections. Studies of Saccharomyces cerevisiae and Candida albicans mutants identify the membrane-spanning subunit of glucan synthase, encoded by the FKS genes, as the molecular target of echinocandins. In vitro, the echinocandins show potent antifungal activity against Candida and Aspergillus species but are much less potent against C. neoformans. In order to examine why C. neoformans cells are less susceptible to echinocandin treatment, we have cloned a homolog of S. cerevisiae FKS1 from C. neoformans. We have developed a generalized method to evaluate the essentiality of genes in Cryptococcus and applied it to the FKS1 gene. The method relies on homologous integrative transformation with a plasmid that can integrate in two orientations, only one of which will disrupt the target gene function. The results of this analysis suggest that the C. neoformans FKS1 gene is essential for viability. The C. neoformans FKS1 sequence is closely related to the FKS1 sequences from other fungal species and appears to be single copy in C. neoformans. Furthermore, amino acid residues known to be critical for echinocandin susceptibility in Saccharomyces are conserved in the C. neoformans FKS1 sequence.

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^* Corresponding author. Mailing address: Department of Infectious Diseases, Merck Research Laboratories, P.O. Box 2000, R80Y-230, Rahway, NJ 07076-0900. Phone: (732) 594-4766. Fax: (732) 594-1399. E-mail: john_thompson{at}merck.com.

Present address: Department of Biological Sciences and Institute for Biomolecular Structure and Function, Hunter College of the City University of New York, New York, NY 10021.

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Journal of Bacteriology, January 1999, p. 444-453, Vol. 181, No. 2
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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