Calcineurin and Aspergillus fumigatus Pathogenesis

描述（由申请人提供）：我的总体假设是钙调磷酸酶介导的信号通路是抗曲霉活性的潜在抗真菌靶点。有大量的数据支持钙调磷酸酶在其他真菌的内稳态和毒力中的作用，我有初步的分子、体外和体内数据支持烟曲霉的研究。在阐明烟曲霉中钙调神经磷酸酶功能方面取得进展的同时，我觉得我需要在实验室得到进一步的支持，以发展我在Madne生物实验室分子真菌学课程中获得的分子技术。我的长期目标是继续我作为一名内科科学家的发展，并成为一名独立的曲霉实验室调查员。我的研究将在杜克大学真菌学研究单位进行，这是一个由美国国立卫生研究院资助的单位，拥有超过75名科学家，在分子真菌学研究方面有着广泛的成功历史。

DESCRIPTION (provided by applicant): 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.