Ras signaling in C. neoformans virulence and development

描述（由申请人提供）：Ras分子是高度保守的信号传导元件，其介导对多种细胞外信号的细胞应答。Ras通路在哺乳动物系统中已被广泛研究，并与许多人类恶性肿瘤的发病机制有关。在微生物中，Ras元件调节包括真菌酵母-菌丝体转变的分化事件。新型隐球菌是一种强大的遗传模型系统，可以用来研究Ras信号在人类病原体中的分子机制。Alspaugh博士先前已经证明，这种生物体中的Ras途径通过MAP激酶途径控制菌丝分化，通过cAMP途径控制琼脂粘附，通过温度依赖性调节肌动蛋白极化来控制高温生长。 在这项提议中，Alspaugh实验室将使用几种无偏倚的遗传学方法来进一步表征C.调节高温生长和细胞分化的新形式。首先，与Ras 1物理相互作用的蛋白质将通过酵母双杂交筛选来鉴定。预计Ras 1信号转导通路的上游和下游编码基因将被确定通过这种方法。此外，Ras蛋白在高温下调节生长的下游组分将通过两种互补的方法来鉴定。Ras 1信号传导的阳性效应子将通过多拷贝抑制分析来鉴定。 将使用在ras 1突变体背景中的随机插入诱变和筛选恢复高温生长来鉴定参与ras 1高温生长控制的抑制或调节蛋白。最后，Alspaugh实验室与杜克大学的其他真菌学实验室合作，正在构建基于C.新人类基因组计划。将使用这些基因微阵列鉴定由Ras 1转录调控的基因。将通过基因破坏和在隐球菌病动物模型中的体外以及体内分析所得突变株来评估通过任何这些方法鉴定的基因的生物学相关性。

DESCRIPTION (provided by applicant): Ras molecules are highly conserved signaling elements that mediate cellular responses to a variety of extracellular signals. Ras pathways have been extensively studied in mammalian systems and implicated in the pathogenesis of many human malignancies. Among microorganisms, Ras elements regulate differentiation events including fungal yeast-mycelium transitions. The basidiomycete Cryptococcus neoformans provides a powerful genetic model system in which to dissect the molecular mechanisms of Ras signaling in a human pathogen. Dr. Alspaugh has previously demonstrated that Ras pathways in this organism control hyphal differentiation through a MAP kinase pathway, agar adherence through a cAMP pathway, and high temperature growth by a temperature-dependent regulation of actin polarization. In this proposal, the Alspaugh laboratory will use several unbiased genetic methods to further characterize the Ras pathways in C. neoformans that regulate high temperature growth and cellular differentiation. First, proteins that physically interact with Ras1 will be identified by a yeast two-hybrid screen. It is expected that genes encoding both upstream and downstream components of the Ras1 signal transduction pathway will be identified by this method. Additionally, downstream components of Ras proteins that regulate growth at elevated temperature will be identified by two complementary approaches. Positive effectors of Ras1 signaling will be identified by multicopy suppressor analysis. Inhibitory or regulatory proteins involved in Ras1 control of high temperature growth will be identified using random insertional mutagenesis in the ras1 mutant background and screening for the restoration of high temperature growth. Finally, in collaboration with other mycology laboratories at Duke University, the Alspaugh lab is constructing gene microarrays based on the C. neoformans genome project. Genes that are transcriptionally regulated by Ras1 will be identified using these gene microarrays. The biological relevance of genes identified by any of these approaches will be assessed by gene disruption and analysis of the resulting mutant strains both in vitro as well as in vivo in animal models of cryptococcosis.