Members of the Ascomycete genus Aspergillus are common animal and plant pathogens. Aspergillus fumigatus causes the majority of Aspergillus human infections, however, inability to identify bona-fide virulence factors has impeded therapeutic advances of this recalcitrant mold. Recently an A. fumigatus mutant, DlaeA, has been characterized that is reduced in virulence. LaeA is a transcriptional regulator of secondary metabolism in the Aspergilli and the A. fumigatus DlaeA mutant is impaired in the production of numerous secondary metabolites including mycotoxins (including gliotoxin, helvolic acid, fumagillin) and spore metabolites. Loss of virulence is associated with loss of gliotoxin levels and associated reduced mortality and growth in a pulmonary murine model, increased susceptibility to macrophage phagocytosis and decreased ability to kill neutrophil cells. The overall goal of this research is to elucidate the mechanisms leading to decreased virulence of the DlaeA mutant with emphasis on identifying the secondary metabolites contributing to pathogenicity.
Specific aims i nclude:
Aim 1. Determine the contribution of loss of gliotoxin production to the DlaeA phenotype. This will be achieved through examination of a gliotoxin mutant, DgliZ, and comparison of this mutant to DlaeA and WT for virulence in pulmonary murine model and macrophage and neutrophil assays.
Aim 2. Identify genes of other mycelial and conidial secondary metabolites involved in A. fumigatus pathogenicity. The method will consist of identifying the genes involved in biosynthesis of mycelial metabolites via microarray analysis in vitro (media) and in vivo (mouse lung). Chemical purification of spore metabolites followed by reverse genetics will be employed to identify encoding genes.
Aim 3. Functional analysis of A. fumigatus secondary metabolites. Genes identified in Aim 2 will be disrupted, mutant chemically defined and examined for virulence as in Aim 1. Advances from this work will result in identification of virulence factors conferring pathogenicity to Aspergillus fumigatus. Knowledge of virulence factors will lead to therapies to help protect the public from infections from this life threatening mold. ? ? ?
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