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.

光滑念珠菌是一种机会性真菌病原体，死亡率高，发病率 由于其对广泛使用的唑类抗真菌药物的高耐药性而增加。光滑 C. 光滑 迅速产生对棘白菌素的耐药性，并可能产生多重耐药性，因此几乎不可能 来治疗。光滑念珠菌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，表现为多种核型和高水平的短核苷酸 菌株间的多态性（SNP）。然而，光滑 C. glabrata 如何促进遗传不稳定性几乎完全是未知数。 未知。所有检查的生物系统中遗传不稳定性的一个主要来源是 DNA 双链断裂 （DSB），介导染色体重排并与高比率的点突变相关 附近地区。因此，光滑 C. glabrata 菌株的染色体重排和 SNP 变异都是 与 DNA DSB 是这种遗传多样性的主要来源一致。事实上，我们的初步研究 表明 C. glabrata 经历 DNA 断裂并产生染色体重排和耐药性 在与宿主细胞相互作用期间发生突变，例如，当驻留在巨噬细胞中时，以及 DSB 的缺失 光滑 C. glabrata 中的修复基因 RAD51 显着增加了小鼠耐药突变体的出现 胃肠道定植模型。该提议基于 C. glabrata 已经进化的假设 DNA 损伤时促进遗传不稳定的机制，要了解这些机制，就需要了解这些机制 了解光滑 C. glabrata 如何生成和处理 DNA DSB 是必要的。在具体目标 1 中，我们建议 使用 DSB 染色质免疫沉淀，然后进行新一代测序 (DSB-ChIP-seq) 和 END- seq（一种高度灵敏、无偏倚的下一代测序技术，用于定量定位 DSB 整个基因组的核苷酸分辨率），以识别光滑 C. glabrata 中容易形成 DSB 的“脆弱”位点，基于 假设这些基因座是最有可能的遗传不稳定性的介质。在具体目标 2 中，我们将使用 DSB-ChIP 和质谱 (DSB-ChIP-MS) 来鉴定介导 DSB 的光滑 C. glabrata 蛋白 交易。在这两个目标中，选定的已识别位点/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 对于了解光滑念珠菌如何促进遗传多样性和进化耐药性至关重要的信息 变种。核型不稳定和异常 DSB 修复也是许多癌症的标志，在这种情况下 了解 DSB 形成和处理的机制有助于开发 针对 DSB 修复机制的有效治疗方法，例如使用 PARP 抑制剂。因此，这个 该提案将首次了解 C. glabrata 中 DSB 的形成和加工，并可能确定其 “阿喀琉斯之踵”，即一种允许其产生遗传多样性但也使其对遗传多样性更加敏感的机制 破坏或损害 DSB 修复的物质。