Candida glabrata Pdr1: Master Regulator of Azole Resistance

描述（由申请人提供）：光滑念珠菌在20世纪90年代作为第二大真菌病原体出现，占酵母分离株的10 - 30%。据报道，侵袭性C。光滑的感染，观察到的最常见的病原体，白色念珠菌的两倍。C. glabrata缺乏与C.白念珠菌（二型性，粘附，水解酶分泌和生物膜形成）;相反，其高患病率和死亡率可能主要归因于其对广泛使用的唑类抗真菌药的内在低水平抗性及其突变获得高水平抗性的能力。我们实验室和其他人的研究显示，在大多数唑类耐药的实验室和临床分离株中，唑类/多药转运蛋白基因CDR 1和PDH 1的表达协同上调，表明这是一种共同的转录因子。事实上，对单个C. glabrata同源的酿酒酵母Pdr 1-Pdr 3，“主调节器”的多药耐药性，确定了推定的功能获得性突变，在多个唑耐药菌株。C. glabrata PDR 1破坏，促进了一种新的PCR为基础的方法，并引入突变的等位基因到敏感菌株提供了进一步的支持，其作用在内在和获得性唑类耐药。此外，微阵列分析显示，Pdr 1介导的基因表达变化超出了CDR 1-PDH 1，可能有助于唑耐药，也改变了C。光叶草毒力在这里，我们提出了三个具体的目标，将表征C。glabrata Pdr 1的深度。这些研究包括：（1）Pdr 1的结构-功能。研究将包括定点诱变，蛋白质结构域相互作用，DNA修复在Pdr 1突变中的作用，二级结构分析和建模Pdr 1进化。(2)CDR 1-PDH 1启动子结构-功能。这些启动子内的调控和核心元件将被确定，和Pdr 1响应元件（PDRE）的序列，多个元素之间的协同性，和进化变异的特点。(3)Pdr 1活性的调节剂。将评估MAP激酶Slt 2、cAMP依赖性蛋白激酶A、组蛋白乙酰转移酶Gcn 5和相关转录因子如Yrm 1在Pdr 1激活中的作用。对“主调节因子”Pdr 1的研究将有助于深入了解C. glabrata唑抗性，并为了解其他病原真菌中类似的抗性机制提供了模型。这对于制定预防或扭转耐药性的有效战略至关重要。光滑念珠菌在20世纪90年代作为第二大真菌病原体出现，占酵母临床分离株的10 - 30%，侵袭性感染的死亡率高达53%。C. glabrata感染可能在很大程度上归因于其对唑类（最广泛使用的抗真菌药组）的内在低水平抗性，以及其突变获得高水平抗性的能力。本论文的研究重点是C. glabrata Pdr 1，唑类耐药的“主调节因子”;他们将深入了解唑类耐药的分子基础，并可能导致预防或逆转耐药的新策略。

DESCRIPTION (provided by applicant): Candida glabrata emerged in the 1990's as the second most important fungal pathogen, representing 10 to 30% of yeast isolates. Mortality rates up 53% have been reported for invasive C. glabrata infection, twice that observed with the most common pathogen, Candida albicans. C. glabrata is deficient in the virulence factors associated with C. albicans (dimorphism, adhesion, hydrolase secretion, and biofilm formation); rather, its high prevalence and mortality may be largely attributed to its intrinsic low-level resistance to widely used azole antifungals and its capacity for mutationally acquired high-level resistance. Studies from our lab and others revealed coordinately upregulated expression of azole/multidrug transporter genes CDR1 and PDH1 in most azole-resistant laboratory and clinical isolates, suggesting a common transcription factor. Indeed, sequence analysis of the single C. glabrata homolog of Saccharomyces cerevisiae Pdr1-Pdr3, "master regulators" of multidrug resistance, identified putative gain-of-function mutations in multiple azole-resistant isolates. C. glabrata PDR1 disruption, facilitated by a novel PCR-based method, and introduction of the mutated allele into a susceptible strain provided further support for its role in both intrinsic and acquired azole resistance. Moreover, microarray analysis has revealed Pdr1-mediated changes in gene expression beyond CDR1-PDH1 that may contribute to azole resistance and also alter C. glabrata virulence. Here we propose three Specific Aims that will characterize C. glabrata Pdr1 in depth. These include: (1) Pdr1 structure-function. Studies will include site-directed mutagenesis, protein domain interaction, the role of DNA repair in Pdr1 mutation, secondary structure analysis, and modeling Pdr1 evolution. (2) CDR1-PDH1 promoter structure-function. Regulatory and core elements within these promoters will be identified, and the Pdr1 response element (PDRE) characterized in terms of sequence, cooperativity between multiple elements, and evolutionary variation. (3) Regulators of Pdr1 activity. The roles in Pdr1 activation of MAP kinase Slt2, cAMP-dependent protein kinase A, histone acetyltransferease Gcn5, and related transcription factors such as Yrm1 will be evaluated. The proposed studies focused on "master regulator" Pdr1 will confer a deep understanding of the molecular basis for C. glabrata azole resistance, and provide a model for understanding similar resistance mechanisms in other pathogenic fungi. This is critical to the development of effective strategies for preventing or reversing resistance. PUBLIC HEALTH RELEVANCE Candida glabrata emerged in the 1990's as the second most important fungal pathogen, representing 10 to 30% of yeast clinical isolates, and with mortality rates up 53% for invasive infection. The high prevalence and mortality of C. glabrata infection may be largely attributed to its intrinsic low-level resistance to azoles, the most widely used group of antifungals, and its capacity for mutationally acquired high-level resistance. The proposed studies focus on C. glabrata Pdr1, "master regulator" of azole resistance; they will confer a deep understanding of the molecular basis for azole resistance, and could lead to novel strategies to prevent or reverse resistance.