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，美国主要的侵袭性真菌病原体。我们的目标是开发一种遗传工具和菌株和数据资源，以促进对基本白色念珠菌基因的理解，从而获得前瞻性的治疗靶点。我们的研究依赖于一种系统的方法来创造部分有缺陷的等位基因，称为“DAmP”等位基因（“mRNA干扰减少丰度”），我们从酿酒酵母遗传学家那里借来。DAmP等位基因是通过用缺乏有效切割和poly-A添加位点的序列取代基因的3'非编码序列而产生的，从而降低mRNA的核输出、稳定性和翻译。在初步研究中，我们成功地将该方法应用于白色念珠菌必需基因CDC3、CDC12、IRE1、SNF1、DBF2和orf19.5376。我们试图通过实现两个特定目标来扩展该方法：(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，所有这些我们都无法用插入突变破坏；(2)通过对DAmP菌株的表型分析，确定这些基因的功能通路。这些目标的完成将提供对白色念珠菌基因功能的新认识，重点关注一组优先考虑包括前瞻性治疗靶点的基因。在可交付成果方面，

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.