Candida glabrata Pdr1: Master Regulator of Azole Resistance

光滑念珠菌 Pdr1：唑耐药性的主要调节因子

• 批准号: 7624574
• 负责人: Thomas D Edlind
• 金额: $ 35.5万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7624574
• 关键词: Adhesions; Alleles; Antifungal Agents; Azole resistance; Azoles; CDR1 gene; Candida albicans; Candida glabrata; Cells; Clinical; Cyclic AMP-Dependent Protein Kinases; DNA Repair; DNA Repair Gene; Development; Elements; Enzymes; Evolution; Gene Expression; Genetic Transcription; High Prevalence; Histones; Homologous Gene; Homology Modeling; Hydrolase; Hyphae; Individual; Infection; Laboratories; Lead; Lipase; Location; Mediating; Methods; Microarray Analysis; Microbial Biofilms; Mitogen-Activated Protein Kinases; Modeling; Modification; Molecular; Multi-Drug Resistance; Mutate; Mutation; Peptide Hydrolases; Phosphorylation Site; Prevalence; Promoter Regions; Proteins; Recruitment Activity; Regulation; Reporting; Resistance; Response Elements; Role; Saccharomyces cerevisiae; Sequence Analysis; Signal Transduction; Site-Directed Mutagenesis; Structure; TATA Box; Tertiary Protein Structure; Testing; Transcription Initiation Site; Variant; Virulence; Virulence Factors; Yeasts; base; cost; deletion analysis; dimorphism; fitness; fungus; gain of function; gain of function mutation; histone modification; mortality; multi drug transporter; mutant; novel; novel strategies; pathogen; prevent; promoter; public health relevance; resistance mechanism; transcription factor

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.

描述（由申请人提供）：念珠菌在1990年代出现为第二重要的真菌病原体，代表10％至30％的酵母菌株。据报道，死亡率上升了53％，用于浸润性曲霉感染，两次观察到最常见的病原体白色念珠菌。 C. glabrata缺乏与白色念珠菌有关的毒力因子（二态，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在很大程度上归因于其内在的低级抗性对广泛使用的硫烷抗真菌抗体以及其突变获得的高级电阻的能力。来自我们实验室和其他人的研究表明，在大多数抗唑的实验室和临床分离株中，Azole/Multidrug Transporter基因CDR1和PDH1的协同上调表达，这表明是常见的转录因子。实际上，对酿酒酵母PDR1-PDR3的单一曲霉同源物的序列分析，多种抗抗性的“主调节剂”，在多种抗烷基抗性分离株中确定了推定的功能增益突变。 C. glabrata PDR1的破坏，通过一种基于PCR的新方法促进，并将突变等位基因引入易感菌株中，为其在固有和获得的Azole耐药性中的作用提供了进一步的支持。此外，微阵列分析揭示了PDR1介导的基因表达的变化以外的CDR1-PDH1，这可能有助于偶氮抗性并改变glabrata毒力。在这里，我们提出了三个特定的目标，这些目标将深入表征C. glabrata Pdr1。其中包括：（1）PDR1结构功能。研究将包括位置定向的诱变，蛋白质结构域的相互作用，DNA修复在PDR1突变中的作用，二级结构分析和建模PDR1演化。 （2）CDR1-PDH1启动子结构功能。将确定这些启动子中的调节和核心元素，并以序列，多个元素之间的合作性和进化变化为特征的PDR1响应元件（PDRE）。 （3）PDR1活性的调节剂。将评估MAP激酶SLT2，CAMP依赖性蛋白激酶A，组蛋白乙酰转移酶GCN5和相关转录因子（如YRM1）中PDR1激活中的作用。拟议的研究集中在“主调节器” PDR1上，将深入理解Glabrata azole抗性的分子基础，并为理解其他致病真菌中类似的耐药机制提供了模型。这对于制定预防或逆转抵抗的有效策略至关重要。公共卫生相关性念珠菌在1990年代出现是第二重要的真菌病原体，占酵母菌临床分离株的10％至30％，死亡率上升了53％。 C. glabrata感染的高患病率和死亡率可能主要归因于其内在的低水平抗性对Azoles，最广泛使用的抗真菌性抗真菌性抗体群，以及其突变获得的高级耐药性的能力。拟议的研究集中于偶氮抗性的“主调节器” C. glabrata Pdr1；他们将深入了解硫代抗性的分子基础，并可能导致预防或逆转抗性的新策略。