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.

光秃念珠菌是一种机会性真菌病原体，死亡率高，发病率高