Identification of CodY-Regulated Factors that Control Sporulation in Clostridioides difficile

艰难梭菌中控制孢子形成的 CodY 调控因子的鉴定

• 批准号: 10751448
• 负责人: Marcos Paulo Monteiro
• 金额: $ 4.77万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751448
• 关键词: Alleles; Amino Acids; Atmosphere; Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; Branched-Chain Amino Acids; Candidate Disease Gene; Cells; Cessation of life; Chemicals; Clostridium difficile; Co-Immunoprecipitations; Colitis; Colonic inflammation; Complement; Data; Diarrhea; Disinfectants; Electrophoretic Mobility Shift Assay; Experimental Designs; Exposure to; Frequencies; Gastrointestinal tract structure; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Germination; Goals; Gram-Positive Bacteria; Growth; Guanosine Triphosphate; Health Care Costs; Human; Infection; Ingestion; Intestines; Investigation; Knowledge; Lead; Life Cycle Stages; Maps; Microscopy; Molecular; Mutate; Nutrient; Nutrient availability; Outcome; Oxygen; Persons; Phenotype; Phototherapy; Production; Proteins; Radiation; Regulation; Repression; Reproduction spores; Role; Signal Pathway; Testing; Toxin; Transcriptional Regulation; Ultraviolet Rays; United States; Variant; Virulence; Work; bile salts; candidate identification; cell motility; detection of nutrient; experimental study; gene function; gut colonization; insight; knock-down; mutant; nutrient deprivation; pathogen; prevent; screening; transcriptome sequencing; transcriptomics; transmission process

PROJECT SUMMARY/ABSTRACT Clostridioides difficile is an anaerobic spore-forming pathogen that causes around 500,000 infections, 13,000 deaths, and healthcare costs of around 1 billion dollars annually in the United States. C. difficile infections cause severe diarrhea and inflammation of the colon. Infection is transmitted by ingestion of C. difficile spores, which can survive oxygen, disinfectants, chemicals, and radiation. After ingestion, spores germinate into vegetative cells within the intestine. These vegetative cells colonize the gastrointestinal tract, generating toxins and forming more spores. Spore formation is triggered by nutritional deprivation of the pathogen in the gastrointestinal tract. Nutrient availability is are sensed in C. difficile by specific transcriptional regulators, such as CodY. CodY is a global transcription regulator that is present in low G + C Gram-positive bacteria and responds to intracellular concentrations of branched-chain amino acids and GTP. Although it is known that C. difficile codY mutants hyper-sporulate, the means by which CodY represses sporulation is not currently understood. Based on the sporulation repression by CodY and its role as a transcriptional regulator, I hypothesize that CodY regulates sporulation by controlling the expression of a sporulation factor(s). In preliminary studies, I performed transcriptomic analyses of codY and wild-type strains, in combination with mapping of CodY-binding sites and transcriptional knockdown. Through these experiments I identified direct CodY-regulated factors that impact sporulation. The top three candidate genes are CDR20291_1334, CDR20291_1851-1853, and CDR20291_2485-2486. To test the hypothesis, I propose to complete these Specific Aims: Aim 1, I will assess the roles of CDR20291_1334, CDR20291_1851-1853, and CDR20291_2485-2486 in sporulation by generating null mutants and subsequently analyzing their contribution to sporulation and transcriptional profiles. Also, I will characterize the basic functions of these genes in C. difficile using growth curves, fluorescent microscopy, and co-immunoprecipitation experiments. In Aim 2, I will compare CodY binding to these target genes on wild-type alleles and variants containing mutated CodY binding sites by electrophoretic mobility shift assay, followed by assessment of the impact of CodY-dependent regulation on sporulation frequency via complementation studies with these genes containing wild-type and mutated CodY boxes. The completion of these aims will identify CodY- regulated genes that control sporulation and uncover their role in sporulation regulation through CodY.

项目摘要/摘要 梭状芽胞杆菌艰难梭菌是一种厌氧孢子形成的病原体，引起约500,000次感染，13,000 在美国，每年的死亡人数和医疗保健费用约为10亿美元。艰难梭菌感染导致 严重的腹泻和结肠炎症。感染是通过摄入艰难梭菌孢子传播的， 可以存活氧气，消毒剂，化学物质和辐射。摄入后，孢子发芽成营养 肠内的细胞。这些营养细胞在胃肠道中定居，产生毒素并形成 更多的孢子。孢子的形成是由胃肠道中病原体的营养剥夺引发的。 特定的转录调节剂（例如Cody）在艰难梭菌中感受到营养的可用性。科迪是一个 在低G + C革兰氏阳性细菌中存在的全局转录调节剂，对细胞内反应 分支链氨基酸和GTP的浓度。尽管众所周知艰难梭菌科迪突变体 超置态，目前尚不了解Cody抑制孢子形成的手段。基于 我假设科迪及其作为转录调节剂的作用，我假设科迪 通过控制孢子孢子因子的表达来调节孢子形成。在初步研究中，我 对Cody和野生型菌株进行了转录组分析，结合了Cody结合的映射 站点和转录敲低。通过这些实验，我确定了直接受CODY调节的因素 影响孢子形成。前三名候选基因是CDR20291_1334，CDR20291_1851-1853和 CDR20291_2485-2486。为了检验假设，我建议完成以下具体目标：AIM 1，我将评估 CDR20291_1334，CDR20291_1851-1853和CDR20291_2485-2486的角色在孢子中生成 无效突变体，随后分析其对孢子形成和转录曲线的贡献。另外，我会的 使用生长曲线，荧光显微镜和 共免疫沉淀实验。在AIM 2中，我将在野生型上比较Cody与这些靶基因的结合 通过电泳迁移率分析，含有突变的Cody结合位点的等位基因和变体，其次是 评估Cody依赖性调节对孢子频率的影响通过互补研究 这些基因包含野生型和突变的Cody盒。这些目标的完成将确定cody- 调控基因控制孢子形成并揭示通过Cody在孢子形成调节中的作用。