SWITCHING AND CANDIDA PATHOGENESIS--A MOLECULAR ANALYSIS

描述（改编自申请人摘要）：C。白色念珠菌开关 自发地、可逆地和以高频率在多个一般 通过菌落形态可区分的表型。 切换具有 对细胞表型的多效性作用，并发生在骨骼肌和 致病种群 表型性状的组合变化 提供C.白色念珠菌的适应性优势，以应对变化， 主持人 使用菌株WO-1中的白色-不透明相转变作为 实验模型，最近证明，开关涉及 精确激活和失活的阶段特异性基因，在 在白色阶段特异性基因WH 11的情况下， 受其启动子中的两个转录激活结构域调控，并且这些 结构域与白色但不与不透明细胞形成相特异性复合物 蛋白提取物 这些结果导致了工作假设， 白色阶段特异性基因由白色阶段特异性激活剂调节， 最近的凝胶阻滞研究表明， 可由白色阶段特异性阻遏物调节。 具体 该提案的目标是：（1）制定一个准确的模型， 参与阶段特异性基因调控的电路; 2）识别 基因位点，并描述了基本开关的一般机制 事件; 3）评估单个阶段特异性基因在 转换表型的发生和4）评估个体的作用 阶段特异性毒力基因。 为了获得一个完整的图片， 涉及的调节事件的调节电路和层次结构 阶段特异性基因调控的程序，我们已经开发了几个 克隆额外的阶段特异性基因的策略。 的推动者 选择的白色和不透明相基因将被功能表征， 使用新开发的海肾荧光素酶生物发光 报告系统，以确定顺式作用序列和调控模式 （阳性和阴性），和凝胶阻滞试验进行的 顺式作用序列和白色或不透明细胞提取物，以鉴定 相特异性复合物。 为了阐明基本的开关事件， 将确定特定阶段的反式作用因子， 阶段特异性基因的表达，其本身是转录的， 监管. 顺式作用序列将被用作探针来筛选一个 反式作用因子基因的表达文库。 假如受规管 因子赋予结合特异性，但不直接结合 顺式作用调节序列，分离策略将涉及 因子的亲和纯化，蛋白质初级测定 序列和基于序列到蛋白质序列的探针设计， 筛查相关基因 个别阶段特定的作用 转换中的基因将通过错误表达的产生来评估 在错误的相位中表达个体相位特异性的突变体， 阶段特异性基因的破坏物，并表征表型 后果 最后，评估角色转换和表达 时相特异性基因在发病机制中，同样的错误表达和无效 将在两种动物模型中研究突变体，一种模型中白色细胞 比不透明细胞更具毒性，并且使用不透明细胞的小鼠模型， 细胞比白色细胞毒性更强。

DESCRIPTION (Adapted from applicant's abstract): C. albicans switches spontaneously, reversibly and at high frequency between a number of general phenotypes distinguishable by colony morphology. Switching has a pleiotropic effect on cellular phenotype and occurs in commensal and pathogenic populations. The combinatorial changes of phenotype traits provide C. albicans an adaptive advantage for responding to changes in the host. Using the white-opaque phase transition in strain WO-1 as an experimental model, it was recently demonstrated that switching involves precise activation and deactivation of phase-specific genes, that in the case of the white phase-specific gene WH11, white-specific transcription is regulated by two transcription activation domains in its promoter, and these domains form phase-specific complexes with white, but not opaque cell protein extract. These results have led to the working hypothesis that white phase-specific genes are regulated by white phase-specific activators, and recent gel retardation studies suggest that opaque phase-specific genes may be regulated by white phase-specific repressors. The specific objectives of this proposal are: 1) to develop an accurate model for the circuitry involved in the regulation of phase-specific genes; 2) to identify the genetic locus and describe the general mechanism of the basic switch event; 3) to assess the roles played by individual phase-specific genes in the genesis of switch phenotypes and 4) to asses the role of individual phase-specific genes in virulence. To obtain a complete picture of the regulatory circuitry and hierarchy of regulatory events involved in the program of phase-specific gene regulation, we have developed several strategies for cloning additional phase-specific genes. The promoters of select white and opaque phase genes will be functionally characterized, using a newly developed Renilla reniformans luciferase bioluminescence reporter system, to identify cis-acting sequences and the mode of regulation (positive and negative), and gel retardation assays carried out with the cis-acting sequences and white or opaque cell extract to identify phase-specific complexes. To elucidate the basic switch event, a phase-specific trans-acting factor will be identified which controls expression of a phase-specific gene and itself is transcriptionally regulated. The cis-acting sequence will be used as a probe to screen an expression library for the trans-acting factor gene. If the regulated factor confers binding specificity but does not directly bind to the cis-acting regulatory sequence, the strategy for isolation will involve affinity purification of the factor, determination of protein primary sequence and the design of probes based on sequence to protein sequence to screen for the gene in question. The role of individual phase-specific genes in switching will be assessed by the generation of mis-expression mutants which express individual phase-specific in the wrong phase and disruptants of the phase-specific genes, and characterize the phenotypic consequences. Finally, to assess the role of switching and the expression of phase specific genes in pathogenesis, the same mis-expression and null mutants will be studied in two animal models, one model in which white cells are more virulent than opaque cells, and a mouse using model in which opaque cells are more virulent than white cells.