DNA Repair Mechanisms in Candida albicans

描述(由申请人提供)： 尽管大量观察表明临床分离株之间存在广泛的基因组差异，但人类病原体白色念珠菌的DNA修复尚未被研究。这种变异与多种基因组改变有关，包括缺失、易位、倍性改变以及染色体或部分染色体的丢失。这些变化与真菌在寄主中的适应、毒力和生长的关系尚不确定，但体外糖利用和抗药性与新倍体有关。同源重组[HR]和非同源末端连接[NHEJ]应该是DNA修复中的重要组成部分，以确保这些基因组改变的保真度。我们用HR的Rad52p和NHEJ途径的Lig4p进行的初步研究表明，这两种蛋白除了在白色念珠菌中提供DNA修复外，还提供生长功能。这一事实以及这种病原体令人惊讶的基因组变异性表明，酿酒酵母和白色念珠菌的DNA修复途径存在差异。为了了解HR和NHEJ在该生物体中的作用，我们提出了四个具体目标。具体目标1包括在两条途径中的每一条中构建新的突变体，NHEJ中的HDF1和HR中的RAD59。在特定的目标2中，我们将完成对PIF1和RRM3的分析，这两种蛋白质是从与Lig4p相互作用的双杂交筛选中鉴定出来的。HR和NHEJ途径蛋白在DNA修复和形态发生中的作用是特定目标3的重点。此外，基因芯片分析将用于确定白念珠菌突变株中上调和下调的基因，并与野生型细胞进行比较。最后，Aim 4旨在评估几个基因缺失突变体在念珠菌病发病机制中的作用。我们建议使用三种念珠菌病小鼠模型来评估毒力，包括侵袭性、口腔和阴道模型，以确定这些基因在毒力中的位置特异性作用。总之，我们的研究将提供关于DNA修复对白念珠菌适应、生长、变异性和毒力的贡献的关键信息。

DESCRIPTION (provided by applicant): DNA repair has not been studied in the human pathogen Candida albicans in spite of numerous observations that suggest extensive genomic variability among clinical isolates. This variability has been associated with a variety of genomic alterations that include, deletions, translocations, ploidy changes, as well as loss of chromosomes or parts of chromosomes. The relationship of these changes to adaptation, virulence, and growth of the fungus in the host is uncertain, but sugar utilization and drug resistance have been correlated in vitro with aneuloploidy. Homologous recombination [HR] and non-homologous end-joining [NHEJ] should be important components in DNA repair to ensure the fidelity of these genomic alterations. Our preliminary studies with Rad52p of the HR and Lig4p of the NHEJ pathways suggest that both proteins provide growth functions in addition to DNA repair in C. albicans. This fact along with the surprising genomic variability of this pathogen suggest differences between the DNA repair pathways of S. cerevisiae and C. albicans. To understand the roles of HR and NHEJ in this organism, we propose four specific aims. Specific aim 1 includes the construction of new mutants in each of the two pathways, HDF1 in NHEJ and RAD59 in HR. In specific aim 2, we will complete our analysis of PIF1 and RRM3, two proteins identified from a two-hybrid screen that interact with Lig4p. The role of HR and NHEJ pathway proteins in DNA repair and morphogenesis is the focus of specific aim 3. In addition, microarray analysis will be used to identify up and down regulated genes in selected mutants of C. albicans when compared to wild type cells. Finally, aim 4 is designed to evaluate the role of several gene deleted mutants in the pathogenesis of candidiasis. We propose to use three mouse models of candidiasis to evaluate virulence, including, invasive, oral, and vaginal models so as to identify a site-specific role of these genes in virulence. In summary, our studies will provide critical information on the contribution of DNA repair to the adaptation, growth, variability and virulence of C. albicans.