Ras signaling in C. neoformans virulence and development

超出提供的空间。RAS分子是高度保守的信号元件，调节细胞对各种细胞外信号的反应。RAS通路在哺乳动物系统中得到了广泛的研究，并与许多人类恶性肿瘤的发病机制有关。在微生物中，RAS元件调节分化事件，包括真菌酵母-菌丝体的转变。新生隐球菌属提供了一个强大的遗传模型系统来研究人类病原体中RAS信号的分子机制。Alspaugh博士已经证明，在这个生物体中，RAS途径通过MAP激酶途径控制菌丝分化，通过cAMP途径控制琼脂黏附，并通过依赖温度调节肌动蛋白极化来控制高温生长。在这项建议中，阿尔斯波实验室将使用几种无偏见的遗传方法来进一步表征新生葡萄球菌中调节高温生长和细胞分化的RAS途径。首先，与RASL物理相互作用的蛋白质将通过酵母双杂交筛选来鉴定。这种方法有望识别编码RASL信号转导通路上游和下游成分的基因。此外，调节高温下生长的RAS蛋白的下游成分将通过两种互补的方法进行鉴定。RASL信号的正效应将通过多拷贝抑制子分析来确定。参与RASL控制高温生长的抑制或调节蛋白将通过在RASL突变背景中的随机插入突变和恢复高温生长的筛选来鉴定。最后，与杜克大学的其他真菌学实验室合作，阿特斯波实验室正在构建基于新生葡萄球菌基因组计划的基因微阵列。受RASL转录调控的基因将使用这些基因微阵列进行识别。通过这些方法中的任何一种方法确定的基因的生物学相关性将通过基因破坏和在体外和体内的隐球菌病动物模型中对所产生的突变菌株的分析来评估。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. 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 Rasl will be identified by a yeast two-hybrid screen. It is expected that genes encoding both upstream and downstream components of the Rasl 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 Rasl signaling will be identified by multicopy suppressor analysis. Inhibitory or regulatory proteins involved in Rasl control of high temperature growth will be identified using random insertional mutagenesis in the rasl mutant background and screening for the restoration of high temperature growth. Finally, in collaboration with other mycology laboratories at Duke University, the Atspaugh lab is constructing gene rnicroarrays based on the C. neoformans genome project. Genes that are transcriptionally regulated by Rasl 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. PERFORMANCE SITE ========================================Section End===========================================