Elucidating mediators of genetic instability in Candida glabrata

阐明光滑念珠菌遗传不稳定性的介质

• 批准号: 10593240
• 负责人: Erika Shor
• 金额: $ 27.74万
• 依托单位: HACKENSACK UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-09 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10593240
• 关键词: Antibodies; Antifungal Agents; Azole resistance; Azoles; CRISPR/Cas technology; Candida; Candida glabrata; Cell division; Cells; ChIP-seq; Chromosome Fragile Sites; Chromosomes; Collaborations; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Sequence; Data Analyses; Data Set; Double Strand Break Repair; Drug resistance; Epitopes; Evolution; Exposure to; G-Quartets; Genes; Genetic; Genetic Polymorphism; Genetic Variation; Genome; Genome Stability; Genomics; Haploidy; Impairment; In Vitro; Incidence; Karyotype; Knowledge; Lesion; Macrophage; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measurement; Mediating; Mediator; Mitosis; Modeling; Multi-Drug Resistance; Mus; Mutation; New York; Nucleotides; Open Reading Frames; Phenotype; Phosphotransferases; Pilot Projects; Point Mutation; Poly(ADP-ribose) Polymerase Inhibitor; Probability; Process; Proteins; Proteomics; Pulsed-Field Gel Electrophoresis; Reporter; Reporting; Resistance; Resolution; Role; S phase; Saccharomyces cerevisiae; Signal Transduction; Site; Source; Structure; Techniques; Testing; Transfer RNA; Universities; Variant; biological systems; chromatin immunoprecipitation; experience; experimental study; fungus; gastrointestinal; gene repair; genetic evolution; genetic variant; homologous recombination; metabolomics; mortality; mutant; next generation sequencing; pathogenic fungus; prevent; resistance frequency; resistance mutation; resistant strain; response; success; therapeutically effective; virtual

Candida glabrata is an opportunistic fungal pathogen associated with high mortality and whose incidence is increasing due to its high frequency of resistance to the widely used azole antifungal class. C. glabrata also rapidly evolves resistance to echinocandins and can become multi-drug resistant and thus virtually impossible to treat. Drug resistance in C. glabrata is acquired via specific genetic variants. C. glabrata is also notable for its remarkable genetic diversity, manifested by a variety of karyotypes and high levels of short nucleotide polymorphisms (SNPs) among strains. However, how C. glabrata facilitates genetic instability is almost entirely unknown. A major source of genetic instability in all examined biological systems are DNA double-strand breaks (DSBs), which mediate chromosome rearrangements and are associated with high rates of point mutations in nearby regions. Thus, both chromosome rearrangements and SNP variation across C. glabrata strains are consistent with DNA DSBs being the major source of this genetic diversity. Indeed, our preliminary studies showed that C. glabrata experiences DNA breaks and develops chromosome rearrangements and drug-resistant mutations during its interaction with host cells, e.g., while residing in macrophages, and that deletion of DSB repair gene RAD51 in C. glabrata significantly increases the emergence of drug-resistant mutants in the mouse gastrointestinal colonization model. This proposal is based on the hypothesis that C. glabrata has evolved mechanisms that facilitate genetic instability upon DNA damage and that to understand these mechanisms it is necessary to understand how C. glabrata generates and processes DNA DSBs. In Specific Aim 1, we propose to use DSB chromatin immunoprecipitation followed by next generation sequencing (DSB-ChIP-seq) and END- seq (a highly sensitive, unbiased next-generation sequencing technique for quantitatively mapping DSBs at nucleotide resolution across the genome) to identify “fragile” loci prone to DSB formation in C. glabrata, based on the hypothesis that these loci are the most likely mediators of genetic instability. In Specific Aim 2, we will use DSB-ChIP followed by mass spectrometry (DSB-ChIP-MS) to identify C. glabrata proteins that mediate DSB transactions. In both Aims, the roles of selected identified loci/genes in DSB formation/processing and genome stability will be validated experimentally. The proposed study will fill a large gap in knowledge and provide information essential for understanding how C. glabrata promotes genetic diversity and evolves drug-resistant variants. Karyotype instability and aberrant DSB repair are also hallmarks of many cancers, and in that context understanding the mechanisms underlying DSB formation and processing has been instrumental in developing effective therapeutic approaches targeting DSB repair mechanisms, e.g., by using PARP inhibitors. Thus, this proposal will provide the first understanding of DSB formation and processing in C. glabrata and may identify its “Achilles’ heel”, i.e., a mechanism that allows it to generate genetic diversity but also makes it more sensitive to agents that disrupt or compromise DSB repair.

念珠菌glabrata是一种与高死亡率相关的机会性真菌病原体，其事件是 由于其对广泛使用的甲唑抗真菌类别的耐药性高频率而增加。 C. glabrata也是如此 迅速发展对echinocandins的抵抗力，并且可以变得多药，因此几乎是不可能的 治疗。 C. glabrata中的耐药性是通过特定的遗传变异获得的。 C. glabrata也因其 显着的遗传多样性，由多种核型和高水平的短核苷酸表现出来 菌株中的多态性（SNP）。但是，C。glabrata如何促进遗传不稳定性几乎完全是 未知。在所有检查的生物系统中，遗传不稳定性的主要来源是DNA双链断裂 （DSB），介导染色体重排，与高点突变相关 附近地区。这是染色体重排和跨C. glabrata菌株的SNP变化 与DNA DSB是这种遗传多样性的主要来源。确实，我们的初步研究 表明C. glabrata会经历DNA断裂并发展染色体重排和耐药性 突变与宿主细胞相互作用时，例如，居住在巨噬细胞中，并缺失DSB 修复基因rad51在glabrata中显着增加了小鼠药物抗药性突变体的出现 胃肠道定植模型。该提议基于以下假设：C。glabrata已进化 促进DNA损害遗传不稳定的机制，并且要理解这些机制 了解C. glabrata如何生成和处理DNA DSB所需。在特定目标1中，我们提出了 使用DSB染色质免疫沉淀，然后进行下一代测序（DSB-CHIP-SEQ）和末端 - SEQ（一种高度敏感的，公正的下一代测序技术，用于定量映射DSB 跨基因组的核苷酸分辨率）以识别基于C. glabrata中DSB形成的“脆弱”基因座，基于C. 关于这些地方是遗传不稳定的介体的假设。在特定目标2中，我们将使用 DSB-CHIP随后进行质谱（DSB-ChIP-MS），以识别介导DSB的glabrata蛋白 交易。在这两个目标中，选定的局部/基因在DSB形成/加工和基因组中的作用 稳定性将通过实验验证。拟议的研究将填补知识的巨大空白，并提供 了解C. glabrata如何促进遗传多样性并发展耐药性的信息至关重要 变体。核型不稳定和异常DSB维修也是许多癌症的标志，在这种情况下 了解DSB形成和处理的基础机制在开发方面起了重要作用 有效的治疗方法针对DSB修复机制，例如使用PARP抑制剂。那，这个 提案将对C. glabrata中的DSB形成和加工提供首次理解，并可能确定其 “阿喀琉斯的脚跟”，即一种使其产生遗传多样性但也使其对其更敏感的机制 破坏或损害DSB修复的代理。