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（PK）在耐药性中的重要性的知识存在很大的差距 致病真菌念珠菌的机制。我们的长期目标是识别药物中的关键激酶 抵抗并利用它们进行新的念珠菌药物开发。我们现在的整体目标 应用是（i）构建一个C. glabrata pk缺失突变库，（ii）系统地筛选突变体 图书馆在抗真菌药物耐药性中的作用，（iii）分析其调节网络，（iv）检查其 在C. glabrata耐药性分离株中的功能。 CNA1是磷酸酶钙调蛋白的催化亚基，是 在甲状腺梭状芽胞杆菌棘齿药物耐受性中必不可少的。通过直接去磷酸化，CNA1激活 转录因子CRZ1随后诱导FKS2（echinocandins的靶标）的表达。在 此外，我们的初步数据表明，CNA1的损失还提高了glabrata的敏感性 三唑（另一种类别的抗真菌药物）。这是通过降低表达和功能的作用 PDR1，C。glabrata三唑耐药性的关键转录因子。进一步分析表明PDR1 实际上，可以将磷酸化，其磷酸化水平随着三唑处理而增加。在一起，这些 调查结果表明，蛋白质磷酸化在抗两者的抗性梭菌机制中很重要 echinocandin和triazole药物，PK作为潜在的治疗靶标，以增强的疗效 这两种抗真菌剂对这种固有的抗性物种。在AIM 1中，我们将构建和验证 使用瞬态CRISPR/CAS9策略，全基因组范围内的圆锥形梭状芽胞杆菌PK缺失突变体。目标 2，我们将检查PK缺失突变体在抗真菌抗真菌耐药性耐药性中的特征。 PK 在耐药性中具有重要作用的候选人将通过竞争性健身和最低限度来确定 抑制浓度（MIC）测定。通过转录分析（RNA-Seq），我们还将探索 这些PK的基因组网络。最后，它们在耐药性中的作用也将在C. glabrata中进行测试 耐药性分离株。提出的研究具有创新性，因为它们独特地关注C的角色。 抗真菌耐药性的glabrata PK。此外，我们的方法是我们第一次创新的 在真菌病原体C. glabrata的临床分离株中生成条形码的C. glabrata PK缺失文库。这 图书馆将为真菌研究界做出重大贡献，因为它适用于体外和 在C. glabrata Biology中对PK功能的体内深度分析。