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.

光滑念珠菌是一种与高死亡率相关的机会性真菌病原体， 由于其对广泛使用的唑类抗真菌剂的高频率耐药性而增加。C.光滑的， 迅速进化出对棘白菌素的耐药性，并可能产生多重耐药性， 治疗C. glabrata是通过特定的遗传变异获得的。C. glabrata也值得注意的是， 显著的遗传多样性，表现为多种核型和高水平的短核苷酸 多态性（SNP）。然而，如何C。glabrata促进遗传不稳定性几乎完全是 未知在所有检测的生物系统中，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，并与高频率的点突变相关， 附近地区。因此，C.光滑菌株是 这与DNA双链断裂是这种遗传多样性的主要来源相一致。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。 在其与宿主细胞相互作用期间发生突变，例如，而DSB的缺失 修复基因RAD 51在C. glabrata显著增加小鼠耐药突变体的出现 胃肠道定植模型。这个建议是基于假设，C。glabrata已经进化 促进DNA损伤后遗传不稳定性的机制，为了理解这些机制， 需要了解C。glabrata产生并加工DNA DSB。具体目标1： 使用DSB染色质免疫沉淀，然后进行下一代测序（DSB-ChIP-seq）和END- seq（一种高灵敏度、无偏倚的下一代测序技术，用于定量定位DSB， 核苷酸分辨率）来鉴定C.光滑，基于 假设这些位点是遗传不稳定性的最可能的介质。在具体目标2中，我们将使用 DSB-ChIP随后质谱法（DSB-ChIP-MS）鉴定C.介导DSB的光滑蛋白 交易在这两个目的中，选择鉴定的基因座/基因在DSB形成/加工和基因组中的作用 将通过实验验证稳定性。这项拟议中的研究将填补知识上的巨大空白，并提供 了解C. glabrata促进遗传多样性并进化出耐药性 变体。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形成和加工的基本机制，对于开发 靶向DSB修复机制的有效治疗方法，例如，通过使用PARP抑制剂。因此，这 该提案将提供对C中DSB形成和处理的初步理解。glabrata和可能识别其 “阿喀琉斯之踵”，即，一种机制，使其能够产生遗传多样性，但也使其对 破坏或损害DSB修复的药物。