New elements of the Target of Rapamycin pathway in Candida albicans

描述(由申请人提供)：这项建议有两个目标：有助于我们理解真菌机会致病菌白念珠菌中Tor信号的生物学，并确定与雷帕霉素类似物协同抗真菌活性的潜在药物靶点。Tor途径促进真核生物的生长和增殖，以响应营养的可获得性和有害应激源的缺乏。在模型酵母和后生动物中，这条通路对许多基本细胞过程的中心作用已经显现出来。在白念珠菌的发病机制中，Tor信号被预测起着至关重要的作用，原因有三。首先，Tor途径共同调节白色念珠菌的形态发生。其次，真菌在宿主吞噬细胞中遇到饥饿条件；第三，宿主免疫系统施加有害压力。Tor途径也很重要，因为它被原核产物雷帕霉素特异性地抑制，为开发真菌特异性雷帕霉素类似物治疗念珠菌感染打开了大门。白色念珠菌和模型酵母的Tor途径之间的部分差异已经变得明显。为此，提出了一种无偏见的正向遗传方法，以发现模式生物中未知的白色念珠菌Tor信号的新成分，并识别可以增强雷帕霉素类似物杀灭活性的小分子抑制的新靶点。我们建议对我们的杂合转座子突变集合进行筛选，因为有了这种试剂，参与Tor信号转座信号的必要基因就可以被识别出来。这种收集的另一个好处是，由杂合子显示的单倍体不足表型在相应的纯合子中更有可能是严重的，使筛查偏向于仅间接与ToR相关的轻微影响的突变。使用这个突变体集合的中试筛选产生了我们两个目标中每个目标的代表：分离出一个在后生动物免疫系统发育过程中发出信号的激酶调节器的同源基因，因为它是雷帕霉素敏感性所必需的。其次，在一个可能的质膜离子泵中发现了一个突变体，它对雷帕霉素高度敏感。这种离子泵没有人类的同源物。其保守的酿酒酵母同系物已被证明被两个不同的小分子抑制，支持可以找到更多抑制剂的想法。鉴定这些基因将有助于我们对白色念珠菌Tor信号的基本了解，并可能为协同抗念珠菌药物的小分子筛选奠定基础。 公共卫生相关性：白色念珠菌是侵袭性真菌感染的最常见原因，即使治疗得当，死亡率也很高。这项工作将确定白念珠菌生长和增殖所需的中央信号通路的新成分，并表征协同抗真菌治疗的潜在药物靶点。

DESCRIPTION (provided by applicant): This proposal has two goals: contribute to our understanding of the biology of Tor signaling in the fungal opportunistic pathogen C. albicans, and identify potential drug targets for synergistic antifungal activity with rapamycin analogs. The Tor pathway promotes growth and proliferation in eukaryotes in response to availability of nutrients and absence of noxious stressors. The centrality of this pathway to many essential cellular processes has emerged from work in model yeasts and in metazoans. In C. albicans pathogenesis, Tor signaling is predicted to play a crucial role for three reasons. Firstly, the Tor pathway co-regulates morphogenesis in C. albicans. Secondly, the fungus encounters starvation conditions in host phagocytes; and thirdly, noxious stresses are imposed by the host immune system. The Tor pathway is also important because it is specifically inhibited by the prokaryotic product rapamycin, opening the door to development of fungal-specific rapamycin analogs for treatment of Candida infections. Partial divergence between the Tor pathway of C. albicans and that of model yeasts has already become apparent. For this reason, an unbiased forward genetic approach is proposed to discover novel components of C. albicans Tor signaling not known from model organisms, and to identify new targets for inhibition by small molecules that can potentiate the cidal activity of rapamycin analogs. We propose a screen of our heterozygous transposon mutant collection because with this reagent, essential genes involved in Tor signaling can be identified. Another advantage of this collection is that haploinsufficiency phenotypes, displayed by heterozygotes, are more likely to be severe in the corresponding homozygotes, biasing the screen against mutations with mild effects only indirectly linked to Tor. A pilot screen using this mutant collection yielded representatives of each of our two goals: a homolog of a kinase regulator, which signals in development of the metazoan immune system, was isolated because it is required for rapamycin sensitivity. Secondly, a mutant in a putative plasma membrane ion pump was found to be exquisitely rapamycin hypersensitive. This ion pump has no human homolog. Its conserved S. cerevisiae homolog has been shown to be inhibited by two different small molecules, supporting the idea that further inhibitors can be found. Characterizing these genes will contribute to our basic understanding of C. albicans Tor signaling and possibly lay the groundwork for a small molecule screen for synergistic candidacidal drugs. PUBLIC HEALTH RELEVANCE: Candida albicans is the most common cause of invasive fungal infections, which even treated appropriately, carry a high mortality. This work will identify new components of a central signaling pathway required for growth and proliferation of C. albicans, and characterize a potential drug target for synergistic antifungal therapy.