Molecular dissection of the genes involved in biosynthesis of polyketide and secondary metabolites of Aspergillus fumigatus and their significance 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. In the previous years, 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. 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. During 2004-2005, we optimized the Agrobacterium tumefaciens mediated transformation (ATMT) system for A. fumigatus. ATMT has proven to be an efficient molecular tool for insertional mutagenesis as well as gene disruption by homologous recombination.? Using the ATMT system, we disrupted the LaeA gene known to be a global regulator for the expression of several important toxins among which gliotoxin is considered to be most important for virulence of A. fumigatus. During 2005-2006, we studied the effect of laeA deletion in the ability of Aspergillus culture supernatant to cause cell detachment and death of EL4 thymoma and MEF cell lines, inhibition of oxidative burst in human PMN, morphogenesis of conidia and virulence in mouse strain 129S. We also deleted the glip gene which encodes the non-ribosomal peptide synthase catalyzing the first step of gliotoxin production. During 2006-2007, we determined that both laeA ko and glip ko strains loose their ability to synthesize gliotoxin and were significantly less virulent that the wild type or reconstituted strains in the mice immunosuppressed with corticosteroids. The reduced virulence in these ko strains was in contrast to works by other groups who used neutropenic mice treated with both cychlophosphamide and corticosteroid.The laeA ko strain, unlike previous reports, produced conidia morphologically indistinguishable from those produced by the wild type strain. In the study of host-pathogen interactions, we found that the neutrophils from CGD patients and normal volunteers were equally efficient in blocking the growth of conidia indicating that neutrophils from both subjects exert an antifungal mechanism independent from the oxidative burst since neutophils from CGD patients are defective in generating superoxide. We determined that the antifungal compound is lactoferrin, a protein secreted during degranulation of neutrophils. The mycelium, in contrast,was effectively inhibited only by neutrophils from normal volunteers and not by neutrophils from CGD patients which, confirmed the previous studies.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Intramural Research (Z01)
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Ibrahim, Ashraf S; Gebremariam, Teclegiorgis; Liu, Mingfu et al. (2008) Bacterial endosymbiosis is widely present among zygomycetes but does not contribute to the pathogenesis of mucormycosis. J Infect Dis 198:1083-90
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
Chang, Yun C; Tsai, Huei-Fung; Karos, Marvin et al. (2004) THTA, a thermotolerance gene of Aspergillus fumigatus. Fungal Genet Biol 41:888-96
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