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.

项目总结/摘要 艰难梭菌是一种厌氧孢子形成病原体，可导致约50万例感染， 死亡和医疗费用每年约为10亿美元。C.艰难梭菌感染导致 严重腹泻和结肠炎。感染是通过摄入C.艰难孢子， 可以在氧气消毒剂化学品和辐射中存活摄入后，孢子萌发成营养体， 肠道内的细胞。这些营养细胞定植在胃肠道，产生毒素， 更多的孢子孢子形成是由胃肠道中病原体的营养剥夺引发的。 养分有效性是在C.通过特定的转录调节因子，如CodY。科迪是一 一种存在于低G + C革兰氏阳性细菌中的全局转录调节因子，对细胞内 支链氨基酸和GTP的浓度。虽然已知C.艰难杆菌codY突变体 对于高孢子形成，CodY抑制孢子形成的手段目前尚不清楚。基于 CodY对孢子形成的抑制作用及其作为转录调节因子的作用，我推测CodY 通过控制孢子形成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学分析，并结合CodY结合位点的定位， 位点和转录敲低。通过这些实验，我确定了直接的CodY调节因子， 影响孢子形成。前三个候选基因是CDR20291_1334、CDR20291_1851-1853和CDR20291_1854。 CDR20291_2485-2486。为了验证假设，我建议完成以下具体目标：目标1，我将评估 CDR20291_1334、CDR20291_1851-1853和CDR20291_2485-2486在孢子形成中的作用， 无效突变体并随后分析它们对孢子形成和转录谱的贡献。我必 描述了这些基因在C.使用生长曲线，荧光显微镜， 免疫共沉淀实验。在目标2中，我将比较CodY与野生型上这些靶基因的结合， 通过电泳迁移率变动分析，检测含有突变CodY结合位点的等位基因和变体，然后 通过互补研究评估CodY依赖性调节对孢子形成频率的影响 这些基因含有野生型和突变的CodY盒。这些目标的完成将确定CodY- 调控基因控制孢子形成，并揭示其在孢子形成调控通过CodY的作用。