Candida glabrata Pdr1: Master Regulator of Azole Resistance

描述(申请人提供)：光滑假丝酵母菌出现在20世纪90年代S作为第二大真菌病原菌，占酵母菌分离物的10%至30%。据报道，侵袭性光滑念珠菌感染的死亡率高达53%，是最常见的白色念珠菌死亡率的两倍。光滑念珠菌缺乏与白色念珠菌相关的毒力因子(二形性、粘附性、水解酶分泌和生物膜形成)；相反，它的高患病率和死亡率在很大程度上可能归因于它对广泛使用的唑类抗真菌药物的内在低水平耐药性和它对突变获得的高水平耐药性的能力。我们实验室和其他实验室的研究发现，在大多数对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CDR1和PDH1的表达协同上调，表明存在共同的转录因子。事实上，对酿酒酵母Pdr1-Pdr3的单一光滑葡萄球菌同源物的序列分析发现，在多个对唑耐药的菌株中，可能存在功能获得突变。一种新的基于聚合酶链式反应的方法促进了光面弯曲霉PDR1的破坏，并将突变的等位基因引入敏感品系，进一步支持了它在内在和获得性唑类耐药性中的作用。此外，微阵列分析揭示了Pdr1介导的CDR1-PDH1以外的基因表达的变化，这可能与唑类耐药性有关，也可能改变光滑念珠菌的毒力。在这里，我们提出了三个具体的目标，将深入表征光肩星天牛Pdr1。这些包括：(1)Pdr1结构-功能。研究将包括定点突变、蛋白质结构域相互作用、DNA修复在Pdr1突变中的作用、二级结构分析和模拟Pdr1进化。(2)CDR1-PDH1启动子的结构与功能。将确定这些启动子中的调控元件和核心元件，并根据序列、多个元件之间的协作性和进化变异来表征Pdr1反应元件(PDRE)。(3)Pdr1活性的调节。将评估MAP激酶SLT2、cAMP依赖的蛋白激酶A、组蛋白乙酰转移酶Gcn5和相关转录因子如Yrm1在Pdr1激活中的作用。对Pdr1“主调控因子”的研究将有助于深入了解光滑假单胞菌耐药的分子基础，并为理解其他病原真菌类似的耐药机制提供一个模型。这对于制定预防或扭转耐药性的有效战略至关重要。与公共卫生相关的光滑假丝酵母菌是20世纪90年代S以来出现的第二大真菌病原菌，占酵母菌临床分离株的10%~30%，侵袭性感染的死亡率高达53%。光滑念珠菌感染的高患病率和高死亡率在很大程度上可能是由于其对唑类药物的内在低水平抗性，以及它对突变获得的高水平抗性的能力。拟议的研究重点放在光泽葡萄球菌Pdr1，它是唑类耐药性的“主要调节器”；它们将使人们深入了解唑类耐药性的分子基础，并可能导致预防或逆转耐药性的新策略。

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.