Molecular dissection of the genes involved in A. fumigatus spore color synthesis and their role in virulence. Aspergillus is one of the most common fungal pathogens affecting neutropenic patients and other types of immunocompromised individuals such as those with Chronic Granulomatous Disease of Childhood. Among a dozen species of Aspergillus reported to cause infection in humans, A. fumigatus is the most common species reported to cause invasive aspergillosis. All Aspergillus species propagate by conidia (spores), which humans encounter daily through inhalation. We have focused our attention on the molecular genetic aspects of conidial pigment biosynthesis since pigment is one of the visible components of the wall that protect conidia. In the previous years we have characterized six developmentally associated genes involved in pentaketide melanin synthesis which are clustered within a 19kb fragment of A. fumigatus genomic DNA. Furthermore, we have shown that the conidial pigment synthetic pathway plays an important role in pathogenesis. In 2001-2002, we identified the product of one of the six clustered genes, AYG1, a novel protein. Ayg1p, shortens the length of the polyketide carbon skeleton from a heptaketide to pentaketide. This year we have purified the Ayg1 protein and identified its reaction mechanism and properties. The purified Ayg1p converted the heptaketide YWA1 to the pentaketide T4HN with release of the deketide acetoacetate, which was confirmed by LC-MS analysis as its o-(2,3,4,5,6-pentafluorobenzyl)oxime. This conversion was strongly inhibited by serine protease inhibitor 3,4-dichloroisocoumarin which modifies serine residue in the catalytic center. Thus, it was concluded that Ayg1p catalyzes the hydrolytic cleavage of C-C bond between naphthalene ring and side chain 1'-carbonyl of YWA1 attcked by hydroxy anion of active center serine residue. Then, o-acetoacetylated Ayg1p is hydrolyzed to release acetoacetate. From the kinetic analysis of Ayg1p reaction, Km for YWA1 and kcat were determined to be 44 um and 4.6 s-1, respectively. Ayg1p showed strict substrate specificity to heptaketide naphthopyrone YWA1 and only 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (ATHN) could serve as a substrate of Ayg1p far less efficiently. ATHN also inhibited strongly by Ayg1p with ki 1.7 um.
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| Sugui, Janyce A; Chang, Yun C; Kwon-Chung, K J (2005) Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: an efficient tool for insertional mutagenesis and targeted gene disruption. Appl Environ Microbiol 71:1798-802 |
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| Tsai, H F; Fujii, I; Watanabe, A et al. (2001) Pentaketide melanin biosynthesis in Aspergillus fumigatus requires chain-length shortening of a heptaketide precursor. J Biol Chem 276:29292-8 |
