Calcineurin and Aspergillus fumigatus Pathogenesis

钙调神经磷酸酶和烟曲霉发病机制

• 批准号: 6816585
• 负责人: WILLIAM J STEINBACH
• 金额: $ 10.97万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6816585
• 关键词: Aspergillus; antifungal agents; biological signal transduction; calcineurin; clinical research; enzyme activity; fluorescent dye /probe; gene expression; laboratory mouse; pathologic process; phosphoprotein phosphatase; polymerase chain reaction; single nucleotide polymorphism; southern blotting; virulence

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.

描述（由申请人提供）：我的总体假设是钙调神经磷酸酶介导的信号通路是抗曲霉活性的潜在抗真菌靶标。有大量的数据支持钙调神经磷酸酶在其他真菌中的稳态和毒力的作用，我有初步的分子，体外和体内数据，支持这项调查在烟曲霉。在阐明钙调神经磷酸酶在A.在研究烟曲霉时，我觉得我需要的是实验室的进一步支持，以发展我的分子技术，超越我在Madne生物实验室的分子真菌学课程中获得的知识。我的长期目标是继续我作为一名医生科学家的发展，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，该单位由NIH资助，拥有超过75名科学家，具有成功的分子真菌学研究的广泛历史。 我将通过使用alcA诱导型启动子来调节钙调神经磷酸酶途径基因的表达来表征钙调神经磷酸酶途径的分子功能，以确定它们是否是必需的。我还将使用钙调磷酸酶抗性突变体，并筛选突变体和A。fumigatus推定同系物。我已经发现钙调磷酸酶抑制剂对A.烟曲霉，我也会测试非免疫抑制类似物。我还将用荧光活性染料表征抗真菌活性的位置，并测量A.将烟曲霉暴露于抗真菌剂以测量作为应激反应防御机制的钙调神经磷酸酶途径。使用一种新的吸入性动物模型，我将测试无毒时，钙调磷酸酶途径的结构与诱导型alcA启动子抑制。我还将测试非免疫抑制性钙调磷酸酶抑制剂类似物，以降低免疫抑制的总水平，从而增加利用钙调磷酸酶途径的总体抗真菌功效。