FUNCTIONAL ANALYSIS OF ESSENTIAL C. ALBICANS GENES

描述(申请人提供)：我们的重点是白色念珠菌，在美国的主要侵袭性真菌病原体。我们的目标是开发一种遗传工具以及菌株和数据资源，以促进对白色念珠菌基本基因的了解，从而达到预期的治疗目标。我们的研究依赖于一种系统化的方法来创造部分有缺陷的等位基因，称为“潮湿”等位基因(“因mRNA扰动而减少的丰度”)，这是我们从酿酒酵母遗传学家那里借来的。DAMP等位基因是通过用缺乏有效切割和PolyA加成位点的序列取代基因的3‘非编码序列而产生的，从而降低了核输出、稳定性和mRNA的翻译。在初步研究中，我们成功地将该方法应用于白色念珠菌的基本基因CDC3、CDC12、IRE1、SNF1、DBF2和orf19.5376。我们试图通过实现两个具体目标来扩展这一方法：(1)创建17个蛋白激酶相关基因和50个细胞壁相关基因的DAMP菌株，所有这些基因都无法通过插入突变中断；以及(2)通过DAMP菌株表型分析将这些基因分配到功能途径。这些目标的完成将提供对白色念珠菌基因功能的新理解，重点放在一组优先包括预期治疗靶点的基因上。在交付成果方面， 该项目将产生新的方法学工具、特征化的菌株收集和数据集，这将使白色念珠菌研究界受益。就总体影响而言，我们预计，我们在这一方法上的成功将促进它在白念珠菌和其他真核病原体中更广泛地实施。 与公共卫生相关：真菌病原体每年在1万多名美国人中导致致命感染，并增加超过26亿美元的医疗费用。需要新药来解决这一健康威胁。我们的项目将开发一种新的方法来了解真菌病原体中的药物靶点。

DESCRIPTION (provided by applicant): Our focus is Candida albicans, the predominant invasive fungal pathogen in the USA. Our goal is to develop a genetic tool and a resource of strains and data that facilitate understanding of essential C. albicans genes, hence prospective therapeutic targets. Our studies rely upon a systematic approach to create partially defective alleles, called "DAmP" alleles ("decreased abundance by mRNA perturbation"), that we have borrowed from S. cerevisiae geneticists. DAmP alleles are created by replacing the 3' noncoding sequence of a gene with sequences that lack efficient cleavage and poly-A addition sites, thus reducing nuclear export, stability, and translation of the mRNA. In pilot studies we have applied the approach successfully to the essential C. albicans genes CDC3, CDC12, IRE1, SNF1, DBF2, and orf19.5376. We seek to extend the approach by implementing two specific aims: (1) to create DAmP strains for 17 protein kinase-related genes and 50 cell wall-related genes, all of which we have been unable to disrupt with insertion mutations; and (2) to assign these genes to functional pathways through DAmP strain phenotypic analysis. Completion of these aims will provide new understanding of C. albicans gene function, focused on a set of genes prioritized to include prospective therapeutic targets. In terms of deliverables, the project will yield new methodological tools, a characterized strain collection, and a dataset that will benefit the C. albicans research community. In terms of overall impact, we foresee that our success with this approach will promote its implementation more broadly in C. albicans and in other eukaryotic pathogens. PUBLIC HEALTH RELEVANCE: Fungal pathogens cause lethal infections in over 10,000 Americans per year and add over $2.6 billion to our health care costs. New drugs are needed to address this health threat. Our project will develop a new method to understand drug targets in fungal pathogens.