Mechanistic analysis and engineering probiotic bacteria with SagA

项目摘要 多重耐药细菌日益成为全球传染病死亡的原因。因为 文明正处于后抗生素时代的边缘，治疗传染病的替代方法需要 被利用。一种特别阴险的细菌病原体，艰难梭菌，在人类肠道中茁壮成长， 在抗生素治疗后释放毒素，减少肠道菌群。C.菌感染 (CDI)可引起大量腹泻和结肠炎，是导致胃肠炎的主要原因之一。 死亡人数CDI很难治疗，因为C.艰难梭菌在抗生素治疗期间可形成休眠孢子 并在治疗结束后导致复发。有证据表明粪便微生物群移植或治疗 益生菌可以预防或治疗CDI。然而，基于益生菌的保护的机制基础 还没有被完全理解。洛克菲勒大学的Hang实验室最近的研究发现， 抗原A（佐贺），一种由肠球菌分泌的假定肽聚糖水解酶 在小鼠中增加宿主对CDI的免疫力。值得注意的是，佐贺在其他细胞中的重组表达 细菌种类也增加了小鼠对CDI的耐受性，暗示佐贺是一种潜在的治疗药物 可以由益生菌递送的试剂。为了了解佐贺活动如何增强主机保护， 将表征佐贺的肽聚糖水解酶活性。此外，佐贺的稳定表达菌株 将被改造成益生菌。最后，将使用稳定的 益生菌-SagA的工程菌株在小鼠模型中。本项目的长期目标是阐明 佐贺的保护机制和工程益生菌稳定表达和分泌佐贺，为未来设计 针对肠道细菌感染的新型疗法。 目的1：佐贺肽聚糖水解酶活性的生化表征 目的2：构建佐贺在益生菌中的稳定表达菌株 目的3：研究SagA-益生菌对艰难梭菌的保护作用

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