Signaling Pathways in Yeast Azole Response

酵母唑反应中的信号通路

• 批准号: 6729061
• 负责人: Thomas D Edlind
• 金额: $ 23.14万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6729061
• 关键词: Candida albicans; Saccharomyces cerevisiae; antifungal agents; azoles; biological signal transduction; calcium flux; drug resistance; fungal genetics; gene expression; microarray technology; protein kinase C; transcription factor; yeasts

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.

描述（由申请人提供）：已大大增加 严重的真菌感染与免疫功能增强的增加一致 艾滋病等条件。有新的发展 抗真菌剂。特别是抑制型熔岩脱甲基酶的唑 因此，麦角固醇生物合成。包括念珠菌在内的大多数酵母菌 白色唱片对唑敏感。但是，其他念珠菌和许多模具 具有本质上低的唑灵敏度。而且，即使在敏感的真菌中 试唑通常缺乏杀真菌活动，因此复发是 常见的。这些可以通过长期使用最小化，但已选择 正常敏感的真菌中的硫代抗性。这些体内限制 与硫唑使用相关的体外相关性：大多数念珠菌株 表现出硫唑的耐受性（在抑制偶氮浓度下的生长后期生长） 阳性突变体在体外高频出现。分子 还已经确定了相关物：ERG11编码羊毛醇的表达 脱甲基酶和多药耐药性（MDR）基因 占空唑暴露后，外排被上调。这些数据表明真菌 以特定方式回应最终降低其功效的叠唑。这 信号通路介导至少一个组成部分的假设 特定信号转导的研究支持“硫唑响应” 抑制剂（STI），与唑EL抑制偶氮耐受性。 Sti-Azole组合具有相当大的治疗潜力。但是， 基本假设尚未进行严格测试，实际真菌性STI 目标和推定的信号通路它们在运行中的作用仍然不确定。 因此，该提案的具体目的是：（1）确定硫磺的耐受性 （ATO）通过RNA表达和遗传分析的基因：（a）代表性性传播疾病 抑制偶氮的耐受性将检查其对它们的影响 白色念珠菌和s中MDR和ERG基因的硫代基因上调 酿酒酵母。 （b）将检查代表性性传播疾病的影响 使用整体的全球酿酒酵母基因表达中偶氮依赖的变化 基因组微阵列； C.白色念珠菌同源物将类似地检查。 （C） 其他调节的基因（除了已经筛选的MDR和ERG基因以外） 通过阵列分析确定的，将检查其在酿酒酵母中的作用 使用破坏和多复制过表达的硫唑灵敏度；平行线 破坏研究将使用选定的白色念珠菌同源物进行。 （2） 识别涉及硫唑公差和ERG上调的信号通路。 钙信号传导在唑耐受性中的作用将进行严格检查 并确定了该途径的ATO目标。同样，PKC的作用 （细胞完整性）将检查途径并确定其ATO靶标。 最后，负责依赖偶氮依赖的上调的信号通路 ERG表达将以ROX1P和HAPLP转录的重点阐明 因素。