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.

项目概要 细胞必须合成核酸来产生用于蛋白质合成的遗传信息，蛋白质合成是必不可少的 所有生命的过程。核酸由含有含氮碱基（核碱基）的核苷酸组成， 决定大分子中的碱基配对并定义遗传密码。核碱基必须是 合成或从环境中回收用于核酸合成，并且细胞能量需求经常 决定使用这些过程中的哪一个。细菌病原体必须合成或挽救核酸 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 代谢是治疗方法的一个有吸引力的目标。脊椎动物的胃肠道定植于 一群合作的微生物，通过消耗肠道来防止入侵病原体的定植 定殖必需营养物质的环境。肠道病原体艰难梭菌感染宿主 胃肠道受到肠道微生物群扰动的影响，是抗生素相关疾病的主要原因 感染。抗生素对肠道微生物群的干扰会改变肠道环境中的营养环境，C. 艰难梭菌必须与宿主和微生物群竞争以获得关键营养物质来定殖并引起疾病。 抗生素治疗后肠道中发生改变的营养物质之一是核碱基，我们假设 艰难梭菌从抗生素扰乱的肠道中挽救核碱基。我们的初步数据表明，艰难梭菌 拥有独特的代谢途径来挽救硫代修饰的尿嘧啶核碱基 4-硫尿嘧啶 (4-TU)，即 存在于脊椎动物的胃肠道中。艰难梭菌可以代谢 4-TU 作为生长的尿嘧啶来源 而不是需要能量的嘧啶生物合成途径。最近，一种酶能够 代谢 4-TU 已从气单胞菌属物种中得到描述，该物种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 的机制尚未明确。 被描述过。我们已经鉴定出两种旁系同源蛋白（CD196_RS03875 和 CD196_RS15345） 艰难梭菌中含有 DUF523 结构域。此外，我们的工作发现 4-TU 具有生长抑制作用 大肠杆菌，缺乏 DUF523 同源物。我们发现了CD196_RS03875，我们将其命名为TudS （硫尿嘧啶脱硫酶）是 4-TU 代谢所必需的，可保护艰难梭菌免受 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性肠道环境中茁壮成长，并且 本提案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 将艰难梭菌中的 4-TU 转化为尿嘧啶，并鉴定对 4- 重要的其他艰难梭菌基因产物 通过创新的转座子筛选进行 TU 代谢。在具体目标 2 中，我们将确定贡献 使用 4-TU 缺陷突变体感染的动物模型研究 4-TU 代谢对艰难梭菌发病机制的影响 TU代谢。这些研究有可能确定一种挽救和解毒的途径。 未经充分研究的非常规核碱基可能有助于重要肠道病原体的发病机制。