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A survey of heterobasidiomycetous yeasts for the presence of the genes homologous to virulence factors of Filobasidiella neoformans, CNLAC1 and CAP59^b

Molecular Microbiology Section, Laboratory of Clinical Investigation, NIAID, Building 10, 11C304, NIH, Bethesda, MD 20892, USA¹
Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands²

Author for correspondence: K. J. Kwon-Chung. Tel:+1 301 496 1602. Fax: +1 301 402 1003. e-mail: june_kwon-chung{at}nih.gov

Among species of the heterobasidiomycetous yeasts, Filobasidiella neoformans is the only serious pathogen that causes fatal infections in both immunocompromised as well as immunocompetent patients. Three phenotypic characteristics, including growth at 37 °C, extracellular polysaccharide capsule and laccase activity, of F. neoformans are known to play major roles in the pathogenicity of the fungus. Several CAP genes involved in polysaccharide capsule formation, as well as the CNLAC1 gene encoding a laccase, have previously been cloned and characterized. To analyse the presence of these Cryptococcus neoformans virulence factors in other heterobasidiomycetous yeasts, numerous species of heterobasidiomycetous yeasts were screened for the presence of laccase activity and a polysaccharide capsule. Species exhibiting laccase activity and possessing a glucuronoxylomannan (GXM) capsule were screened for homologues of both the CAP59 gene and the CNLAC1 gene of F. neoformans. Southern blots of genomic DNA from GXM capsule-producing species exhibited no discernible hybridization to the CAP59 DNA sequence except for the two varieties of F. neoformans and Cryptococcus podzolicus. Although discernible, the hybridization band observed with the DNA of C. podzolicus was faint. Oligonucleotide primers constructed using the CAP59 gene sequence also failed to yield PCR products from DNAs of these yeasts except for the two varieties of F. neoformans. These results, coupled with the absence of a CAP59 homologue in the database, suggested the CAP59 gene to be unique to F. neoformans. C. podzolicus was the only species besides F. neoformans that possessed a capsule and expressed strong laccase activity on various media containing phenolic compounds. A CNLAC1 homologue was isolated from C. podzolicus while it was not detected in the species producing beige to faint tan colonies on media with phenolic compounds. Compared to the CNLAC1 sequence of four serotypes of F. neoformans, the CNLAC1 homologue of C. podzolicus showed the highest homology to that of serotype B/C strains and the lowest homology to that of serotype A strains.

Keywords: Cryptococcus neoformans, virulence gene homologues, basidiomycetous yeasts

Abbreviations: DOPA, 3,4-dihydroxyphenylalanine; GXM, glucuronoxylomannan; ITS, internal spacer region

^b The GenBank accession numbers for the sequences determined in this work are AF337642, L22866, AF337643, AF337644 and AF337645.

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