Clostridioides difficile nucleobase scavenging in the competitive gut environment

竞争性肠道环境中艰难梭菌核碱基清除

• 批准号: 10677923
• 负责人: Matthew Munneke
• 金额: $ 3.3万
• 依托单位: VANDERBILT 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 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677923
• 关键词: 16S ribosomal RNA sequencing; 4-thiouracil; Aeromonas; Animal Model; Antibiotic Therapy; Antibiotics; Base Pairing; Cells; Cessation of life; Clostridium difficile; Communities; Data; Diarrhea; Disease; Drug Metabolic Detoxication; Enterobacteriaceae; Environment; Enzymes; Escherichia coli; Family; Gastrointestinal tract structure; Genes; Genetic Code; Growth; Health Care Costs; Health care facility; High-Throughput Nucleotide Sequencing; Histopathology; Homologous Gene; Immune system; Infection; Invaded; Life; Mass Spectrum Analysis; Mediating; Metabolic; Metabolic Pathway; Metabolism; Modeling; Molecular; Mus; Names; Nucleic Acids; Nucleotides; Nutrient; Nutritional; Pathogenesis; Pathway interactions; Process; Protein Biosynthesis; Proteins; Pseudomembranous Colitis; Pyrimidine; Reporting; Reproduction spores; Role; Site-Directed Mutagenesis; Source; Testing; Therapeutic Intervention; Thiouracil; Toxic Megacolon; Toxic effect; Toxin; United States; Uracil; Work; acute care; antibiotic-associated diarrhea; base; colonization resistance; competitive environment; enteric pathogen; experimental study; feeding; gastrointestinal epithelium; gene product; genetic information; gut microbiota; innovation; macromolecule; member; microbiota; microorganism; mutant; new therapeutic target; nucleobase; pathogen; pathogenic bacteria; prevent; stressor; targeted treatment

PROJECT SUMMARY Cells must synthesize nucleic acids to create genetic information that is used for protein synthesis, an essential process for all life. Nucleic acids are composed of nucleotides containing a nitrogenous base (nucleobase) that dictates base-pairing in the macromolecule and defines the genetic code. Nucleobases must be either synthesized or salvaged from the environment for nucleic acid synthesis, and cellular energy demands often dictate which of these processes is used. Bacterial pathogens must synthesize or salvage nucleic acids for optimal growth and survival during infection, often through pathways that differ from the host, making nucleobase metabolism an attractive target for therapeutic approaches. The vertebrate gastrointestinal tract is colonized by a cooperative group of microorganisms that prevent colonization by invading pathogens by depleting the gut environment of essential nutrients for colonization. The enteric pathogen Clostridioides difficile infects the host gastrointestinal tract upon perturbation of the gut microbiota and is the leading cause of antibiotic-associated infections. Antibiotic perturbation of the gut microbiota alters the nutrient milieu in the gut environment, and C. difficile must compete with the host and microbiota to obtain critical nutrients to colonize and cause disease. Amongst the nutrients altered in the gut following antibiotic treatment are nucleobases, and we hypothesize that C. difficile salvages nucleobases from the antibiotic perturbed gut. Our preliminary data indicate that C. difficile possesses a unique metabolic pathway to salvage a thio-modified uracil nucleobase, 4-thiouracil (4-TU), that is present in the vertebrate gastrointestinal tract. C. difficile can metabolize 4-TU as a uracil source for growth instead of the energetically demanding pyrimidine biosynthetic pathway. Recently, an enzyme capable of metabolizing 4-TU has been described from an Aeromonas species, representing a member of a large family of enzymes containing a DUF523 domain. However, the mechanism by which C. difficile metabolizes 4-TU has not been described. We have identified that two paralogous proteins (CD196_RS03875 and CD196_RS15345) contain a DUF523 domain in C. difficile. Furthermore, our work has uncovered that 4-TU is growth inhibitory to Escherichia coli, which lacks a DUF523 homolog. We discovered that CD196_RS03875 which we named TudS (thiouracil desulfurase), is required for 4-TU metabolism and protects C. difficile from 4-TU mediated toxicity. We hypothesize that 4-TU metabolism enables C. difficile to thrive in the competitive gut environment, and experiments in this proposal will test this hypothesis. In Specific Aim 1, we will define the molecular mechanism by which TudS converts 4-TU to uracil in C. difficile and identify other C. difficile gene products important for 4- TU metabolism through an innovative transposon screen. In Specific Aim 2, we will determine the contribution of 4-TU metabolism to C. difficile pathogenesis using an animal model of infection with mutants defective in 4- TU metabolism. These studies have the potential to define a pathway for salvage and detoxification of an understudied, unconventional nucleobase that may contribute to the pathogenesis of an important gut pathogen.

项目总结 细胞必须合成核酸来创造用于蛋白质合成的遗传信息，这是一种必不可少的 所有生命的过程。核酸是由含有含氮碱基(碱基)的核苷酸组成的 决定了大分子中的碱基配对，并定义了遗传密码。碱基对必须是 合成的或从环境中回收的用于核酸合成的，细胞能量需求通常 指定使用这些过程中的哪一个。细菌病原体必须合成或回收核酸以 在感染期间的最佳生长和存活，通常通过与宿主不同的途径，使核碱基 新陈代谢是治疗方法的一个有吸引力的目标.脊椎动物的胃肠道由 一组合作的微生物，通过耗尽肠道来防止通过入侵病原体来定居 为定居提供必要营养的环境。肠道致病菌艰难梭状芽胞杆菌感染宿主 肠道微生物区系紊乱的胃肠道，是抗生素相关疾病的主要原因 感染。肠道微生物区系的抗生素扰动改变了肠道环境中的营养环境，而C. 艰难梭菌必须与宿主和微生物区系竞争，以获得定居和致病的关键营养物质。 在抗生素治疗后肠道中改变的营养物质中有核苷酸碱基，我们假设 艰难梭菌从抗生素扰乱的肠道中抢救出碱基。我们的初步数据显示艰难梭菌 拥有一条独特的代谢途径来挽救硫代修饰的尿嘧啶核苷酸碱基4-硫氧尿嘧啶(4-TU)，即 存在于脊椎动物的胃肠道中。艰难梭菌可代谢4-TU作为尿嘧啶来源 而不是能量要求很高的嘧啶生物合成途径。最近，一种能够 代谢4-TU已从一种气单胞菌物种中描述出来，它代表了一个大家族的成员 含有DUF523结构域的酶。然而，艰难梭菌代谢4-TU的机制还没有 都被描述过。我们已经鉴定出两个同源蛋白(CD196_RS03875和CD196_RS15345)。 艰难梭菌中含有DUF523结构域。此外，我们的工作还发现4-TU对细胞生长有抑制作用。 大肠杆菌，缺少DUF523同源基因。我们发现CD196_RS03875我们命名为TudS (硫尿嘧啶脱硫酶)，是4-TU代谢所必需的，并保护艰难梭菌免受4-TU介导的毒性。 我们假设4-TU代谢使艰难梭菌能够在竞争激烈的肠道环境中茁壮成长，并且 这一提议中的实验将检验这一假设。在具体目标1中，我们将定义分子机制 通过TudS在艰难梭菌中将4-TU转化为尿嘧啶，并鉴定对4-TU重要的其他艰难梭菌基因产物. TU新陈代谢通过创新的转座子筛选。在具体目标2中，我们将确定贡献 4-TU代谢对艰难梭菌致病作用的研究 你的新陈代谢。这些研究有可能确定一条抢救和解毒的途径 未被研究的，非传统的碱基，可能有助于一种重要的肠道病原体的发病机制。