Signaling Pathways in Yeast Azole Response

描述（由申请人提供）： 严重的真菌感染与免疫功能低下 如艾滋病。同时，新的 抗真菌剂。具体地，抑制羊毛甾醇脱甲基酶的唑类化合物 并因此导致麦角甾醇生物合成。大多数酵母菌种，包括念珠菌 白色念珠菌是唑敏感的。然而，其他念珠菌属物种和许多霉菌 具有固有的低唑敏感性。此外，即使在敏感的真菌中， 唑类通常缺乏杀真菌活性，因此， 共同这些可以通过长期使用来最小化，但这已经选择了 在正常敏感真菌中的唑类抗性。这些体内限制 与唑类药物使用相关的体外相关性：大多数念珠菌分离株 表现出唑类耐受性（抑制唑类浓度下的拖尾生长） 并且唑耐受突变体在体外以高频率出现。分子 相关性也已被鉴定：编码羊毛甾醇的ERG 11的表达 脱甲基酶和多药耐药（MDR）基因负责唑 在唑暴露后外排被上调。这些数据表明真菌 对唑类药物产生特异性反应，最终降低其疗效。的 假设信号通路介导至少一个组分， “唑类反应”得到特异性信号转导研究的支持 抑制剂（STI），其与唑类组合抑制唑类耐受性。 STI-唑组合具有相当大的治疗潜力。但 潜在的假设尚未得到严格的检验，实际的真菌性传播感染 靶点和它们发挥作用的假定信号通路仍然不确定。 因此，本提案的具体目的是：（1）鉴定唑类药物耐受性 (ATO)通过RNA表达和遗传分析的基因：（a）代表性STI 将检测抑制唑耐受性的药物对 唑依赖性上调C.白色念珠菌和S 啤酒。(b)将审查代表性的性传播感染对 唑依赖的全球S.酿酒酵母基因表达 基因组微阵列; C.将类似地检查白色念珠菌同系物。（c）第（1）款 其他调节基因（已筛选的MDR和ERG基因除外） 通过阵列分析确定将检查它们在S.酿酒酵母 使用破坏和多拷贝过表达的唑敏感性;平行 将对选定的C进行破坏研究。白色念珠菌同系物（二） 确定参与唑耐受性和ERG上调的信号通路。 钙信号传导在唑类药物耐受性中的作用将被严格检验 并确定了该途径的ATO靶点。同样，PKC的作用 (cell完整性）途径，并确定其ATO目标。 最后，负责吡咯依赖性上调的信号通路 ERG表达将重点阐述Rox 1 p和Haplp转录 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.