Mechanistic analysis and engineering probiotic bacteria with SagA

使用 SagA 进行机理分析和改造益生菌

• 批准号: 9920591
• 负责人: Charles W Hespen
• 金额: $ 6.74万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-16 至 2022-04-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9920591
• 关键词: Aftercare; Animals; Antibiotic Therapy; Antibiotics; Antigens; Bacteria; Bacterial Infections; Biochemical; Caenorhabditis elegans; Cell Wall; Chromosomal Insertion; Chromosomes; Civilization; Clostridium difficile; Colitis; Communicable Diseases; Diarrhea; Engineered Probiotics; Engineering; Enteral; Enterococcus faecium; Future; Gastroenteritis; Genes; Goals; Human; Hydrolase; Immunity; In Vitro; Infection; Lactobacillus plantarum; Length; Methods; Modeling; Multiple Bacterial Drug Resistance; Mus; N-Acetylmuramoyl-L-alanine Amidase; Peptidoglycan; Probiotics; Recombinants; Recurrence; Relapse; Reproduction spores; Research; Structure; Therapeutic; Therapeutic Agents; Toxin; Universities; Work; antibiotic-associated diarrhea; base; commensal bacteria; commensal microbes; design; enteric pathogen; fecal transplantation; gut microbiota; lactic acid bacteria; mortality; mouse model; novel therapeutics; pathogenic bacteria; prevent; probiotic therapy

Project Summary Multidrug-resistant bacteria are increasingly the cause of infectious disease fatalities worldwide. Because civilization is at the precipice of a post-antibiotic era, alternative methods to treat infectious diseases need to be utilized. One especially insidious bacterial pathogen, Clostridium difficile, thrives in the human gut and releases its toxin after antibiotic treatment diminishes the commensal intestinal microbiota. C. difficile infection (CDI) can cause profuse diarrhea and colitis and is one of the leading causes of gastroenteritis related fatalities. CDI can be difficult to treat, as C. difficile can form dormant spores during treatment with antibiotics and cause relapse after treatment has ended. There is evidence fecal microbiota transplantation or treatments with probiotic bacteria can prevent or treat CDI. However, the mechanistic basis for probiotic-based protection is not fully understood. Recent work from the Hang Lab at Rockefeller University has identified secreted antigen A (SagA), a putative peptidoglycan hydrolase secreted by the commensal bacterium Enterococcus faecium that increased host immunity to CDI in mice. Remarkably, recombinant expression of SagA in other bacterial species also increased mouse tolerance against CDI implicating SagA as a potential therapeutic agent that can be delivered by a probiotic. In order to understand how SagA activity increases host protection, peptidoglycan hydrolase activity of SagA will be characterized. Additionally, stable expression strains of SagA will be engineered in probiotic bacteria. Finally, host protection against CDI will be investigated using the stably engineered strains of probiotic-SagA in a mouse model. The long-term goals of this project are to elucidate the protective mechanism of SagA and engineer probiotics to stably express and secrete SagA for future design of novel therapeutics against enteric bacterial infections. Aim 1: Biochemical characterization of SagA peptidoglycan hydrolase activity Aim 2: Engineer stable expression strains of SagA in probiotic bacteria Aim 3: Investigate protection of SagA-probiotic against Clostridium difficile

项目摘要 多重耐药细菌正日益成为全球传染病死亡的原因。因为 文明正处在后抗生素时代的边缘，治疗传染病的替代方法需要 被利用。一种特别隐蔽的细菌病原体，艰难梭菌，在人类肠道和 在抗生素治疗减少共生肠道微生物区系后释放其毒素。艰难梭菌感染 (CDI)可引起大量腹泻和结肠炎，是引起胃肠炎的主要原因之一 死亡事故。CDI可能很难治疗，因为艰难梭菌在抗生素治疗期间会形成休眠孢子。 并在治疗结束后导致复发。有证据表明粪便微生物区系移植或治疗 用益生菌可以预防或治疗CDI。然而，益生菌保护的机制基础 还没有完全被理解。洛克菲勒大学Hang实验室最近的研究发现， A抗原(SAGA)，由共生菌肠球菌分泌的一种可能的肽聚糖水解酶 提高小鼠对CDI的宿主免疫力的粪便。值得注意的是，SAGA在其他组织中的重组表达 细菌种类也增加了小鼠对CDI的耐受性，这意味着SAGA是一种潜在的治疗方法 一种可以通过益生菌输送的制剂。为了了解SAGA活动如何增强宿主保护， 将对SAGA的肽聚糖水解酶活性进行表征。此外，SAGA的稳定表达菌株 将在益生菌中进行改造。最后，针对CDI的主机保护将使用 小鼠模型中的益生菌工程菌株。这个项目的长期目标是阐明 SAGA和工程益生菌的保护机制为未来的设计稳定表达和分泌SAGA 治疗肠道细菌感染的新疗法。 目的1：佐贺多肽多糖水解酶活性的生化特性 目的2：在益生菌中稳定表达SAGA的工程菌 目的3：研究佐贺益生菌对艰难梭菌的保护作用