Signaling Pathways in Yeast Azole Response

描述(由申请人提供)： 严重的真菌感染与免疫低下的增加不谋而合 艾滋病等病症。同时也有新的发展 抗真菌药物。具体地说，抑制羊毛甾醇脱甲基酶的唑类化合物 因此麦角固醇的生物合成。包括假丝酵母在内的大多数酵母菌 白色念珠菌对唑类药物敏感。然而，其他念珠菌和许多霉菌 天生对唑类药物的敏感度低。此外，即使是在敏感的真菌中 唑类通常缺乏杀菌活性，因此复发是 很普通。可以通过长期使用将其降至最低，但这已被选择为 正常敏感真菌中的唑类耐药性。这些在体内的限制 与使用唑类药物有体外相关性：大多数念珠菌分离株 表现出对唑类的耐受性(在抑制性唑类浓度下的拖尾生长) 在体外，耐药突变株出现频率较高。分子 还确定了相关因素：编码羊毛甾醇的ERG11的表达 与唑类相关的去甲基酶和多药耐药基因的研究 在使用唑类药物后，外排被上调。这些数据表明，真菌 以特定的方式对最终降低疗效的氮唑类药物做出反应。这个 假设信号通路至少调节这一过程的一个组成部分 与特定信号转导相关的研究支持“唑类反应” 抑制剂(STI)，与氮唑类药物联合使用可抑制唑类耐药。 STI-唑类药物组合具有相当大的治疗潜力。然而， 潜在的假设还没有得到严格的检验，而实际的真菌STI 靶点和它们发挥作用的假定信号通路仍未确定。 因此，这项建议的具体目标是：(1)确定对唑类的耐受性 (ATO)基因的RNA表达和遗传分析：(A)具有代表性的STI 抑制唑类耐受性的药物将被检测其对 唑依赖对白念珠菌和S耐药基因和ERG基因上调的研究 酿酒。(B)将检查典型的性传播感染对以下方面的影响 应用全菌技术研究全球酿酒酵母基因表达的唑依赖性变化 基因组微阵列；白色念珠菌的同源物将进行类似的检查。(C) 其他调控基因(除已筛选的MDR和ERG基因外) 将检查它们在酿酒酵母中的作用 使用干扰和多拷贝过度表达的唑类敏感性；平行 将对选定的白色念珠菌同源物进行干扰研究。(2) 确定与唑耐受和ERG上调有关的信号通路。 将严格研究钙信号在唑类耐药中的作用。 确定了这条通路的ATO靶点。同样，PKC的作用 (细胞完整性)途径将被检查并确定其ATO靶标。 最后，负责唑依赖上调的信号通路。 ERG的表达将被阐明，重点是Rox1p和Haplp转录 各种因素。

DESCRIPTION (provided by applicant): There has been a significant increase in serious fungal infections coincident with the increase in immunocompromised conditions such as AIDS. There has been a parallel development of new antifungal agents. In particular, azoles that inhibit lanosterol demethylase and hence ergosterol biosynthesis. Most yeast species including Candida albicans are azole-sensitive. However, other Candida species and many molds have intrinsically low azole sensitivity. Moreover, even in sensitive fungi azoles generally lack fungicidal activity, and consequently recurrences are common. These can be minimized by long-term use, but this has selected for azole resistance in normally sensitive fungi. These in vivo limitations associated with azole use have in vitro correlates: most Candida isolates exhibit azole tolerance (trailing growth at inhibitory azole concentrations) and azole tolerant mutants arise at high frequency in vitro. Molecular correlates have also been identified: expression of ERG11 encoding lanosterol demethylase and of multidrug resistance (MDR) genes responsible for azole efflux are upregulated following azole exposure. These data suggest that fungi respond in specific ways to azoles that ultimately reduce their efficacy. The hypothesis that signalling pathways mediate at least one component of this 'azole response' is supported by studies with specific signal transduction inhibitors (STIs), which in combination with azoles inhibit azole tolerance. STI-azole combinations have considerable therapeutic potential. However, the underlying hypothesis has not been rigorously tested, and the actual fungal STI targets and the putative signalling pathways they function in remain undefined. Thus, the Specific Aims of this proposal are to: (1) Identify azole tolerance (ATO) genes by RNA expression and genetic analysis: (a) Representative STIs that inhibit azole tolerance will be examined for their effects on azole-dependent upregulation of MDR and ERG genes in C. albicans and S cerevisiae. (b) Representative STIs will be examined for their effects on azole-dependent changes in global S. cerevisiae gene expression using whole genome microarrays; C. albicans homologs will be similarly examined. (c) Additional regulated genes (other than the MDR and ERG genes already screened) identified by array analysis will be examined for their role in S. cerevisiae azole sensitivity using disruption and multcopy overexpression; parallel disruption studies will be done with selected C. albicans homologs. (2) Identify signalling pathways involved in azole tolerance and ERG upregulation. The role of calcium signalling in azole tolerance will be rigorously examined and the ATO targets of this pathway identified. Similarly, the role of the PKC (cell integrity) pathway will be examined and its ATO targets identified. Finally, the signalling pathway responsible for azole-dependent upregulation of ERG expression will be elucidated with a focus on Rox1p and Haplp transcription factors.