Functional and transcriptome analyses of protein kinases in Candida glabrata antifungal drug resistance

光滑念珠菌抗真菌药物耐药性中蛋白激酶的功能和转录组分析

• 批准号: 10643423
• 负责人: Bao Gia Vu
• 金额: $ 19.44万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2023-09-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10643423
• 关键词: Address; Amphotericin B; Antifungal Agents; Antifungal Therapy; Antimicrobial Resistance; Applied Research; Area; Auxins; Azoles; Bar Codes; Basic Science; Biological Assay; Biology; CRISPR/Cas technology; Candida; Candida glabrata; Catalytic Domain; Characteristics; Clinical; Clinical Trials; Communicable Diseases; Communities; Data; Development; Disseminated candidiasis; Drug Targeting; Drug Tolerance; Drug resistance; Exhibits; Fluconazole; Frequencies; Fungal Drug Resistance; Future; Gene Expression Profiling; Genetic; Genomics; Goals; In Vitro; Infection; Knowledge; Libraries; Link; Medicine; Metabolism; Minimum Inhibitory Concentration measurement; Mission; Multi-Drug Resistance; National Institute of Allergy and Infectious Disease; Pathway Analysis; Pharmaceutical Preparations; Pharmacotherapy; Phosphorylation; Phosphotransferases; Polyenes; Predisposition; Protein Analysis; Protein Dephosphorylation; Protein Dynamics; Protein Kinase; Proteins; Public Health; Regulation; Research; Resistance; Resistance profile; Role; Saccharomyces cerevisiae; System; Testing; Time; Triazoles; Work; Yeasts; calcineurin phosphatase; deletion library; drug development; drug sensitivity; fitness; genetic analysis; genetic resource; genome-wide; human pathogen; in vivo; innovation; insight; kinase inhibitor; mutant; novel; pathogenic fungus; preservation; prevent; protein function; resistance mechanism; therapeutic target; transcription factor; transcriptome; transcriptome sequencing

There is a significant gap in knowledge concerning the importance of protein kinases (PKs) in drug resistance mechanisms of the pathogenic fungus Candida glabrata. Our long-term goal is to identify key kinases in drug resistance and exploit them for novel Candida drug development. Our overall objectives in the present application are to (i) construct a C. glabrata PK deletion mutant library, (ii) systemically screen mutants in the library for their roles in antifungal drug resistance, (iii) analyze their regulation networks, and (iv) examine their functions in C. glabrata drug resistance isolates. Cna1, the catalytic subunit of the phosphatase calcineurin, is essential in C. glabrata echinocandin drug tolerance. Through direct dephosphorylation, Cna1 activates the transcription factor Crz1 which subsequently induces the expression of Fks2 (the target of echinocandins). In addition, our preliminary data have indicated that loss of CNA1 also enhances C. glabrata susceptibility to triazoles (a different class of antifungal drug). This works through a reduction in expression and function of Pdr1, the key transcription factor in C. glabrata triazole drug resistance. Further analysis indicate that Pdr1 can, in fact, be phosphorylated and its phosphorylation level increases with triazole treatment. Together, these findings indicate that protein phosphorylation is important in C. glabrata mechanisms of resistance against both echinocandin and triazole drugs, and PKs present as potential therapeutic targets for enhancing the efficacy of these two antifungal agents against this inherently resistant species. In Aim 1, we will construct and validate a genome-wide barcoded set of C. glabrata PK deletion mutants using a transient CRISPR/Cas9 strategy. In Aim 2, we will examine the characteristics of PK deletion mutants in C. glabrata antifungal drug resistance. PK candidates that have important roles in drug resistance will be identified by competitive fitness and minimum inhibitory concentration (MIC) assays. With transcriptional profiling (RNA-seq), we will also explore the genomic networks of these PKs. Finally, their roles in drug resistance will also be tested among C. glabrata drug resistance isolates. The proposed studies are innovative as they uniquely focus on the roles of C. glabrata PKs in antifungal drug resistance. Furthermore, our approach is innovative as we will for the first time generate a barcoded C. glabrata PK deletion library in a clinical isolate of the fungal pathogen C. glabrata. This library will be a significant contribution to the fungal research community, since it is suitable for both in vitro and in vivo future in-depth analyses of PK functions in C. glabrata biology.

关于蛋白激酶（PKs）在耐药性中的重要性，在知识上存在显著差距 致病真菌光滑念珠菌的机制。我们的长期目标是确定药物中的关键激酶， 并利用它们进行新型念珠菌药物开发。我们目前的总体目标 应用程序是（i）构建一个C。glabrata PK缺失突变体文库，（ii）系统地筛选glabrata PK缺失突变体文库中的突变体， 库中的抗真菌药物耐药性的作用，（iii）分析其调控网络，（iv）检查其 函数在C. glabrata耐药分离株。Cna 1是磷酸酶钙调磷酸酶的催化亚基， 在C.光滑棘白菌素耐药性。通过直接去磷酸化，Cna 1激活了 转录因子Crz 1，随后诱导Fks 2（棘白菌素的靶标）的表达。在 此外，我们的初步数据表明，CNA 1的缺失也增强了C.光滑的易感性 三唑类（另一类抗真菌药物）。这是通过减少表达和功能来实现的。 Pdr 1是C.光滑三唑耐药。进一步分析表明，Pdr 1 事实上，可以被磷酸化，并且其磷酸化水平随着三唑处理而增加。所有这些 研究结果表明蛋白磷酸化在C. glabrata对这两种药物的抗性机制 棘白菌素和三唑类药物，以及PKs作为潜在的治疗靶点， 这两种抗真菌剂对抗这种固有的耐药物种。在目标1中，我们将构建并验证一个 C.全基因组条形码集本发明涉及使用瞬时CRISPR/Cas9策略的glabrata PK缺失突变体。在Aim中 2、我们将研究C. PK缺失突变体的特征。光滑型抗真菌药物耐药性。PK 在耐药性中具有重要作用的候选者将通过竞争适应度和最小 抑制浓度（MIC）测定。通过转录谱分析（RNA-seq），我们还将探索 这些PKs的基因组网络。最后，它们在耐药性中的作用也将在C.光滑 耐药分离株。所提出的研究是创新的，因为他们独特地关注C的作用。 光滑型PK在抗真菌药物耐药性中的作用。此外，我们的方法是创新的，因为我们将首次 生成条形码C. glabrata PK缺失文库中的一个临床分离的真菌病原体C。光滑的这 库将是一个重大贡献的真菌研究界，因为它是适合在体外和 在C.光果草生物学