IDENTIFICATION OF A POLYKETIDE SYNTHASE GENE (PKSP) OF ASPERGILLUS-FUMIGATUS INVOLVED IN CONIDIAL PIGMENT BIOSYNTHESIS AND VIRULENCE

Aspergillus fumigatus is an important pathogen of the immunocompromise d host causing pneumonia and invasive disseminated disease with high m ortality. Previously, we identified a mutant strain (white, W) lacking conidial. pigmentation and, in addition, the conidia showed a smooth surface morphology, whereas wild-type (WT) conidia are grey-green and have a typical ornamentation. W conidia appeared to be less protected against killing by the host defence, e.g., were more susceptible to ox idants in vitro and more efficiently damaged by human monocytes in vit ro than WT conidia. When compared to the WT, the W mutant strain showe d reduced virulence in a murine animal model. Genetic analysis suggest ed that the W mutant carried a single mutation which caused all of the observed phenotypes. Here, we report the construction of a genomic co smid library of A. fumigatus and its use for complementation of the W mutant. Transformation of the W mutant was facilitated by co-transform ation with plasmid pHELP1 carrying the autonomously replicating amal s equence of A. nidulans which also increased the transformation efficie ncy of A. fumigatus by a factor of 10. Using this cosmid library a put ative polyketide synthase gene, designated pksP (polyketide synthase i nvolved in pigment biosynthesis) was isolated. The pksP gene has a siz e of 6660 bp. pksP consists of five exons separated by short (47-73 bp ) introns. Its deduced open reading frame is composed of 2146 amino ac ids. The pksP gene complemented both the white phenotype and the surfa ce morphology of the W mutant conidia to wild type. Whereas W mutant c onidia caused a strong reactive oxygen species (ROS) release by polymo rphonuclear leukocytes, the ability of pksP-complemented W mutant coni dia to stimulate ROS release was significantly reduced and comparable to that of WT conidia. In addition, the complemented strains showed re stored virulence in a mouse model.