My global hypothesis is that the calcineurin-mediated signaling pathway is a potential antifungal target for anti-Aspergillus activity. There are extensive data to support the role of calcineurin in homeostasis and virulence in other fungi, and I have preliminary molecular, in vitro, and in vivo data that support this investigation in Aspergillus fumigatus. While making progress towards elucidating calcineurin function in A. fumigatus, I felt what I needed was further support in the laboratory to develop my molecular techniques beyond what I acquired in the Molecular Mycology course at the Madne Biology Laboratory. My long-term goal is to continue my development as a physician scientist and become an independent Aspergillus laboratory investigator. My research will be performed at the Duke University Mycology Research Unit, an NIH-funded unit with over 75 scientists and an extensive history of successful molecular mycology research. I will characterize the molecular function of the calcineurin pathway by using the alcA inducible promoter to regulate expression of calcineurin pathway genes to determine if they are essential. I will also use calcineurin inhibitor-resistant mutants and screen for nucleotide polymorphisms between the mutants and A. fumigatus putative homologs. I have found the calcineurin inhibitors have inherent in vitro antifungal activity against A. fumigatus, and I will also test non-immunosuppressive analogs. I will also characterize the location of the antifungal activity with fluorescent viability dyes, and measure calcineurin phosphatase activity after A. fumigatus is exposed to antifungals to gauge the calcineurin pathway as a stress-response defense mechanism. Using a novel inhalational animal model I will test for avirulence when the calcineurin pathway constructs are repressed with the inducible alcA promoter. I will also test the non-immunosuppressive calcineurin inhibitor analogs to decrease the total level of immunosuppression and therefore increase the overall antifungal efficacy of exploiting the calcineurin pathway.

K08AI061149

2004-08-01

2010-04-30