A Genetic Approach to Virulence in C. albicans

描述(申请人提供)：白色念珠菌是世界上最常见的真菌病原体，但对其毒力机制的了解有限，特别是与细菌病原体的毒力机制相比。对学术前沿认识的匮乏与缺乏安全、有效的临床诊断和治疗工具有关。正向遗传筛选，如签名标记突变，一直是识别细菌主要毒力途径的基础。然而，由于二倍体基因组的实验限制和缺乏完整的交配周期，这种筛查还没有在白念珠菌中进行。我最近开发了减轻这些障碍的方法和试剂。这项建议的目标是对白念珠菌的毒力进行第一次大规模的遗传分析，以识别和研究这种生物致病的关键决定因素。为了实现这些目标，我有以下具体目标：(1)构建白色念珠菌纯合子干扰突变体的签名标签敲除文库；(2)在播散性念珠菌病的小鼠感染模型中对毒力突变体进行遗传筛选；(3)通过对致病周期中特定步骤的二次筛选，表征突变体的毒力缺陷；以及(4)对选定的特殊感兴趣的突变体进行详细的细胞生物学、分子生物学和生化分析。这项工作的结果将加强我们对真菌发病机制的理解，并确定抗真菌治疗的新靶点。

DESCRIPTION (provided by applicant): Candida albicans is the most common fungal pathogen worldwide and yet there is limited understanding of its virulence mechanisms, particularly in comparison to those of bacterial pathogens. This poverty of understanding on the academic front is associated with a dearth of safe, effective clinical diagnostic and therapeutic tools. Forward genetic screens such as signature tagged mutagenesis have been fundamental in identifying the major virulence pathways in bacteria. Such screens have not been performed in C. albicans, however, because of experimental limitations imposed by a diploid genome and the absence of a complete mating cycle. I have recently developed methods and reagents that mitigate these obstacles. The goal of this proposal is to perform the first large-scale genetic analysis of virulence in C. albicans in order to identify and study the key determinants of pathogenesis in this organism. To accomplish these goals, I have the following specific aims: (1) Construct a signature-tagged knockout library of C. albicans homozygous disruption mutants; (2) Perform a genetic screen for virulence mutants in a murine infection model of disseminated Candidiasis; (3) Characterize the virulence defects of the mutants through secondary screens of specific steps in the pathogenesis cycle; and (4) Carry out detailed cell biological, molecular biological, and biochemical analysis of selected mutants of special interest. Results from this work should enhance our understanding of fungal pathogenesis and identify novel targets for antifungal therapy.