Mechanisms and consequences of human milk oligosaccharide growth and bile stress across diverse strains of the potential therapeutic bacterium, Akkermansia muciniphila.

目前的范例认为，粘液阿克曼氏菌是人类肠道的有益成员 微生物组建立在对生理多样性和机械活动的不完全理解的基础上。 在整个谱系中，正如所有以前的工作都集中在一种描述的菌株上一样。我们的长期目标是帮助 开发阿克曼西亚的有针对性的治疗用途，要么通过刺激内源菌株 益生菌或通过给予特定菌株作为益生菌。此应用程序的总体目标是 鉴定猪瘟病毒基因组差异株的分子机制和免疫原性 在人乳寡糖(HMO)上生长的人相关阿克曼氏菌和(II)在 胆汁。这项工作的中心假设是，与人类相关的阿克曼西亚人进化出了不同的生长 HMO和胆汁应激反应在依赖菌株中塑造其免疫原性潜力的效率 举止。这项工作的基本原理是，如果我们要使用阿克曼西亚进行治疗 代谢紊乱或其他胃肠道疾病，那么我们需要设计生物知情的治疗方法 促进或引入精选菌株以实现最佳健康结果的战略。中心假设将是 测试通过追求两个特定的目标：1)确定分子机制和免疫原性 HMO上生长的阿克曼原虫；2)鉴定胆汁抵抗机制和免疫原性 阿克曼草属植物生长在胆汁中。在第一个目标下，目标基因表达研究结合在一起 通过克隆和功能分析，将用于鉴定参与植物生长的糖苷水解酶 HMO在三个基因组不同的阿克曼西亚分离物中的分布。同时，有针对性的代谢组学分析将 用于量化HMO消耗量和发酵最终产品。最后，共培养实验与 HMO培养的阿克曼西亚细胞和人类上皮细胞将被用来测量细菌结合效率 以及上皮细胞的免疫反应。对于第二个目标，转录组和蛋白质组图谱， 结合靶向代谢组学(即胆汁酸成分和微生物胞外多糖) 将被用来表征这三个阿克曼氏菌分离株的胆汁应激反应。与AIM 1类似，co- 胆汁培养阿克曼氏菌株和人上皮细胞的培养实验 细菌结合效率和宿主免疫反应。这项申请中提出的研究是 创新是因为它代表着对现状的实质性偏离，通过提供对 阿克曼草谱系的生理多样性和分子机制。拟议的研究 意义重大，因为它将有助于定义整个世系的生理格局，从而打开新的 推广或引入精选阿克马尼亚菌株的生物知情治疗策略的地平线 以获得最佳的健康结果。

The current paradigm that Akkermansia muciniphila is a beneficial member of the human gut microbiome is based on an incomplete understanding of the physiological diversity and mechanistic activity across the lineage, as all previous work has focused on one described strain. The long-term goal is to help develop targeted, therapeutic uses of Akkermansia, either through stimulating endogenous strains with prebiotics or by administering specific strains as probiotics. The overall objectives for this application are to characterize the molecular mechanisms and immunogenic properties of genomically diverse strains of human-associated Akkermansia grown (i) on human milk oligosaccharides (HMO) and (ii) in the presence of bile. The central hypothesis of this work is that human-associated Akkermansia have evolved different growth efficiencies on HMO and bile stress responses that shape their immunogenic potential in a strain dependent manner. The rationale for this work is that if we are to use Akkermansia for the therapeutic treatment of metabolic disorders or other gastrointestinal diseases, then we need to design biologically informed treatment strategies that promote or introduce select strains for optimal health outcomes. The central hypothesis will be tested by pursuing two specific aims: 1) Identify the molecular mechanisms and immunogenic properties of Akkermansia grown on HMO; 2) Identify the bile resistance mechanisms and immunogenic properties of Akkermansia grown in the presence of bile. Under the first aim, targeted gene expression studies coupled with cloning and functional assays will be used to identify glycoside hydrolase enzymes involved in growth on HMO in three genomically diverse Akkermansia isolates. Concurrently, targeted metabolomics analyses will be used to quantify HMO consumption and fermentation end products. Lastly, co-culture experiments with HMO grown Akkermansia cells and human epithelial cells will be used to measure bacterial binding efficiency and the immune response of the epithelial cells. For the second aim, transcriptomic and proteomic profiling, coupled with targeted metabolomics (i.e. bile acid composition and microbial extracellular polysaccharides) will be used to characterize the bile stress response of the three Akkermansia isolates. Similar to aim 1, co- culture experiments with bile grown Akkermansia strains and human epithelial cells be used to measure bacterial binding efficiency and the host immune response. The research proposed in this application is innovative because it represents a substantive departure from the status quo by providing insights into the physiological diversity and molecular mechanisms across the Akkermansia lineage. The proposed research is significant because it will help define the physiological landscape across the lineage, thereby opening new horizons for biologically informed treatment strategies that promote or introduce select Akkermansia strains for optimal health outcomes.