Calcineurin and Aspergillus fumigatus Pathogenesis

钙调神经磷酸酶和烟曲霉发病机制

基本信息

批准号：
7225274
负责人：
WILLIAM J STEINBACH
金额：
$ 12.05万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
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/7225274
关键词：
A, Calcineurin Affect Animal Model Antifungal Agents Antifungal Therapy Aspergillosis Aspergillus Aspergillus fumigatus Biology Blood specimen Calcineurin Calcineurin Pathway Calcineurin inhibitor Calmodulin Carbon Class Clinical Cyclosporine Data Databases Defense Mechanisms Development Dyes Funding Genes Genetic Polymorphism Goals Homeostasis Homologous Gene Immunosuppression Immunosuppressive Agents In Vitro Incidence Invasive Investigation Laboratories Location Measures Mediating Methods Microscopic Modality Modeling Molecular Mus Nucleotides Organ Transplantation Pathogenesis Pathway interactions Patients Phosphoric Monoester Hydrolases Physicians Recording of previous events Research Research Personnel Resistance Role Scientist Signal Pathway Source Standards of Weights and Measures Stress Techniques Testing The science of Mycology Therapeutic immunosuppression Transplant Recipients Treatment Protocols Universities Virulence analog base biological adaptation to stress calcineurin phosphatase fungus in vivo inhibitor/antagonist mortality mutant novel promoter response transcription factor

项目摘要

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点。有大量数据支持钙调神经磷酸酶在其他真菌体内平衡和毒力中的作用，并且我有初步的分子、体外和体内数据支持烟曲霉的这项研究。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，我觉得我需要实验室的进一步支持，以发展我的分子技术，超越我在马德恩生物学实验室分子真菌学课程中获得的技术。我的长期目标是继续发展成为一名医学科学家，并成为一名独立的曲霉菌实验室研究者。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，在分子真菌学研究方面拥有丰富的成功历史。我将通过使用 alcA 诱导型启动子调节钙调磷酸酶途径基因的表达来表征钙调磷酸酶途径的分子功能，以确定它们是否是必需的。我还将使用钙调神经磷酸酶抑制剂抗性突变体并筛选突变体和烟曲霉推定同源物之间的核苷酸多态性。我发现钙调磷酸酶抑制剂对烟曲霉具有固有的体外抗真菌活性，我还将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，并在烟曲霉暴露于抗真菌剂后测量钙调磷酸酶活性，以测量作为应激反应防御机制的钙调磷酸酶途径。使用新型吸入动物模型，我将测试当钙调神经磷酸酶途径构建体被诱导型 alcA 启动子抑制时的无毒力。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，从而提高利用钙调磷酸酶途径的总体抗真菌功效。