FUNCTIONAL ANALYSIS OF ESSENTIAL C. ALBICANS GENES

描述（由申请人提供）：我们的重点是白色念珠菌，在美国的主要侵入性真菌病原体。我们的目标是开发一种遗传工具和一种菌株和数据资源，以促进对必需C。白念珠菌的基因，因此有前景的治疗目标。我们的研究依赖于一种系统的方法来创建部分缺陷的等位基因，称为“DAmP”等位基因（“通过mRNA扰动减少丰度”），我们从S.酿酒遗传学家通过用缺乏有效切割和聚腺苷酸添加位点的序列替换基因的3'非编码序列，从而降低mRNA的核输出、稳定性和翻译，产生DAmP等位基因。在试点研究中，我们已经成功地将该方法应用于基本C。白色念珠菌基因CDC 3、CDC 12、IRE 1、SNF 1、DBF 2和orf19.5376。我们试图通过实现两个具体目标来扩展该方法：（1）为17个蛋白激酶相关基因和50个细胞壁相关基因创建DAmP菌株，所有这些基因我们都无法用插入突变破坏;（2）通过DAmP菌株表型分析将这些基因分配到功能途径。这些目标的实现将使人们对C.白念珠菌基因功能，集中在一组基因优先，包括前瞻性的治疗目标。在可交付成果方面， 该项目将产生新的方法学工具，一个特征化的菌株收集和一个数据集，这将有利于C。白色念珠菌研究社区。就整体影响而言，我们预见，我们在这一方法上的成功将促进其在C.白色念珠菌和其他真核病原体。 公共卫生相关性：真菌病原体每年在超过10，000名美国人中引起致命感染，并为我们的医疗保健成本增加超过26亿美元。需要新的药物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标。

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.