SWITCHING AND CANDIDA PATHOGENESIS--A MOLECULAR ANALYSIS

描述(摘自申请者摘要)：白色念珠菌开关 自发、可逆和高频之间的若干一般 通过菌落形态可区分的表型。交换有一个 对细胞表型的多效性影响，并出现在共生和 致病种群。表型性状的组合变化 为白念珠菌提供一种适应优势，以应对 主持人。利用菌株WO-1的白色-不透明相变作为 实验模型，最近证明了转换涉及到 特定阶段基因的精确激活和失活，即在 在白色阶段特异基因WH11的情况下，白色特异转录 受其启动子中的两个转录激活结构域调节，这些 结构域与白色细胞而不是不透明细胞形成相特定的络合物 蛋白质提取物。这些结果导致了工作假说 白色阶段特定的基因由白色阶段特定的激活物调控， 最近的凝胶延迟研究表明，不透明的阶段特异性基因 可能受白色阶段特异性抑制物的调节。具体的 这项建议的目标是：1)开发一个准确的模型 参与调控特定阶段基因的电路；2)识别 并描述了基本开关的一般机制 事件；3)评估单个阶段特定基因在 开关表型的起源和4)评价个体的作用 毒力中的阶段特异性基因。要全面了解 涉及的监管电路和监管事件的层级 阶段特异性基因调控程序，我们已经开发了几个 克隆更多特定阶段基因的策略。的推动者 将对选定的白色和不透明相基因进行功能表征， 利用新开发的肾形雷尼拉荧光素酶生物发光 报告人制度，识别顺式作用序列和调控模式 (阳性和阴性)，并用 顺式作用序列和白色或不透明细胞提取液鉴定 特定于相的络合物。要阐明基本切换事件，请使用 将确定特定阶段的反式作用因子，以控制 特定阶段基因的表达，其本身是转录的 受监管的。顺式作用序列将被用作探针来筛选 反式作用因子基因的表达文库。如果受监管的 因子赋予结合特异性，但不直接与 顺式作用调控序列，隔离策略将涉及 因子的亲和纯化及蛋白质原初含量的测定 序列和基于序列到蛋白质序列的探针的设计 筛选出有问题的基因。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 在错误的阶段表达个体特定阶段的突变体和 相特定基因的干扰物，并表征表型 后果。最后，评估转换的作用和表达 在发病机制中的阶段特异性基因中，相同的错误表达和空 突变将在两个动物模型中进行研究，其中一个模型中的白细胞 比不透明细胞毒力更强，以及使用不透明细胞模型的小鼠 细胞比白细胞的毒性更强。

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.