METAGENOMIC ENGINEERING OF PROBIOTIC BACTERIA TO IMPROVE INTESTINAL COLONIZATION

描述(由申请人提供) 摘要：人体肠道微生物区系是一个遗传多样性的微生物群落，对宿主起着至关重要的作用，包括碳水化合物代谢、维生素生产、调节免疫系统、促进上皮屏障功能和排斥病原体。微生物区系的破坏会导致宿主的一系列病理变化，益生菌已经成为治疗或预防这种微生物失调的潜在解决方案。益生菌治疗包括消耗一种或几种活微生物，当给予适当浓度时，这些活微生物会给人类宿主带来好处。我们提出了一种新的合成生物学方法来解决目前面临的最关键的益生菌治疗效果障碍，即益生菌无法永久和稳定地整合到已建立的人类微生物区系中。我们假设(1)天然的人类微生物区系，无论是在发育过程中还是在稳定的时候，都会编码一系列基因，从而能够稳定地整合到一个已建立的群落中；(2)这些特征可以被工程改造成具有假定的益生菌特性的生物，提高它们的肠道定植潜力和适合性。因此，我们将在功能上展望完整的人类微生物区系和定义的肠道微生物培养集合，寻找促进一组候选益生菌整合(统称为整合增强片段或IEF)到哺乳动物宿主稳定微生物区的基因和操纵子，研究已识别基因的机制，并竞争和组合优化最佳整合片段，以选择和工程增强型整合表型。我们还将在小鼠肠道疾病模型中将我们增强的益生菌与传统益生菌进行比较，以确认其有效性。为了实现这些目标，我们将在功能元基因组学、下一代测序和生物质畜牧业方面开发和应用尖端技术创新。因此，我们提案的一个主要预期成果是一种新的平台技术，用于将增强健康的特性工程到假定的益生菌中，促进哺乳动物宿主中发达和稳定的微生物群的整合和持久性。我们将应用这一平台来设计下一代益生菌菌株，使其能够增强整合到已建立的微生物区系中的能力，并有可能快速部署治疗人类胃肠道疾病的治疗方法。 公共卫生相关性：正在考虑通过益生菌治疗来调节人类胃肠道微生物区系，以治疗一些胃肠道疾病。这项提议旨在解决目前推定的益生菌菌株部署的最关键障碍之一，即它们无法长期整合到宿主微生物中。我们将开发新的技术来发现和设计基因元素，使候选益生菌菌株能够稳定地整合到现有的微生物区系中，从而有可能 快速部署用于治疗人类胃肠道疾病。

DESCRIPTION (Provided by the applicant) Abstract: The human intestinal microbiota is a genetically diverse community of microorganisms that performs critical functions for the host, including carbohydrate metabolism, vitamin production, modulation of the immune system, promotion of epithelial barrier function, and exclusion of pathogens. Disruption of the microbiota can lead to a number of pathologies in the host, and probiotics have emerged as a potential solution for treating or preventing such dysbioses. Probiotic treatments consist of the consumption of a strain or several strains of live microorganisms that confer benefits on their human hosts when administered in adequate concentrations. We propose a novel synthetic biology approach to address the most critical barrier currently facing probiotic therapeutic efficacy, namely the inability of probiotic bacteriato permanently and stably integrate into established human microbiota. We hypothesize that (1) native human microbiota, either during development or when stabilized, encode a repertoire of genes which would enable stable integration into an established community and (2) these traits can be engineered into organisms with putative probiotic properties, improving their intestinal colonization potential and fitness. Accordingly, we will functionally prospect complete human microbiota and defined gut microbial culture collections for genes and operons facilitating integration (collectively referred to as integration-enhancing fragments or IEFs) of a set of candidate probiotics into stable microbiota of mammalian hosts, investigate the mechanisms of the identified genes, and compete and combinatorially optimize the best IEFs to select and engineer enhanced integration phenotypes. We will also confirm the efficacy of our enhanced probiotics by comparing them to traditional probiotics in a mouse model for intestinal disease. To achieve these goals, we will develop and apply cutting- edge technological innovations in functional metagenomics, next-generation sequencing and gnotobiotic husbandry. Hence, a major anticipated deliverable of our proposal is a novel platform technology for engineering fitness-enhancing properties into putative probiotic bacteria, promoting integration and persistence in a developed and stable microbiota in a mammalian host. We will apply this platform to engineer next-generation probiotic strains with an enhanced capacity for integration into an established microbiota, with the potential for rapid therapeutic deployment for the treatment of human gastrointestinal diseases. Public Health Relevance: Modulation of the human gastrointestinal microbiota through probiotic therapy is being considered for the treatment of a number of gastrointestinal disorders. This proposal aims to address one of the most critical barriers to the deployment of current putative probiotic strains, namely their inability for long-term integration into the host microbioa. We will develop novel technologies to discover and engineer genetic elements which enable candidate probiotic strains to stably integrate into established microbiota, with the potential for rapid deployment for the treatment of human gastrointestinal disease.