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Infection and Immunity, March 2001, p. 1781-1794, Vol. 69, No. 3
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.3.1781-1794.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Molecular Cloning and Characterization of WdPKS1, a Gene Involved in Dihydroxynaphthalene Melanin Biosynthesis and Virulence in Wangiella (Exophiala) dermatitidis

Bin Feng,1 Xu Wang,1 Melinda Hauser,2 Sarah Kaufmann,2 Simone Jentsch,3 Gerhard Haase,3 Jeffery M. Becker,2 and Paul J. Szaniszlo1,*

Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 787121; Microbiology Department, University of Tennessee, Knoxville, Tennessee 379192; and Institute of Medical Microbiology, University Hospital RWTH Aachen, Aachen, Germany3

Received 21 September 2000/Returned for modification 11 October 2000/Accepted 17 November 2000

1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of Wangiella dermatitidis, an agent of phaeohyphomycosis in humans. To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene, WdPKS1, in W. dermatitidis. The resulting wdpks1Delta disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intact WdPKS1 gene was then successfully recovered from two plasmids. The WdPKS1 gene was also isolated independently by complementation of the mel3 mutation in an albino mutant of W. dermatitidis using a cosmid library. Sequence analysis substantiated that WdPKS1 encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a beta -ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all wdpks1Delta strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in a wdpks1Delta strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.

* Corresponding author. Mailing address: Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, TX 78712. Phone: (512) 471-3384. Fax: (512) 471-7088. E-mail: pjszaniszlo{at}mail.utexas.edu.

Infection and Immunity, March 2001, p. 1781-1794, Vol. 69, No. 3
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.3.1781-1794.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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