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

目前认为嗜粘蛋白阿克曼氏菌是人类肠道的有益成员 微生物组是基于对生理多样性和机械活性的不完全理解 在整个谱系中，因为所有先前的工作都集中在一个描述的菌株上。长期目标是帮助 开发阿克曼氏菌的靶向治疗用途，或者通过刺激内源性菌株， 益生元或通过施用特定菌株作为益生菌。此应用程序的总体目标是 表征基因组多样性菌株的分子机制和免疫原性， 人相关阿克曼氏菌（i）在人乳低聚糖（HMO）上生长，和（ii）在 胆汁这项工作的中心假设是，人类相关的阿克曼氏菌已经进化出不同的生长 对HMO和胆汁应激反应的效率，在菌株依赖性中形成其免疫原性潜力， 方式这项工作的基本原理是，如果我们要使用阿克曼菌来治疗 代谢紊乱或其他胃肠道疾病，那么我们需要设计生物学上知情的治疗方法， 促进或引入选择菌株以获得最佳健康结果的战略。核心假设是 通过追求两个特定目标进行测试：1）确定免疫原性的分子机制和免疫原性特性， 在HMO上生长的阿克曼氏菌; 2）鉴定 阿克曼氏菌在胆汁中生长。在第一个目标下，靶向基因表达研究与 通过克隆和功能测定，将用于鉴定参与生长的糖苷水解酶， 三种基因组多样的阿克曼氏分离株中的HMO。同时，有针对性的代谢组学分析将 用于量化HMO消耗和发酵终产物。最后，共培养实验， HMO生长的阿克曼氏细胞和人上皮细胞将用于测量细菌结合效率 以及上皮细胞的免疫反应。对于第二个目标，转录组学和蛋白质组学分析， 结合靶向代谢组学（即胆汁酸组成和微生物胞外多糖） 将用于表征三种阿克曼氏菌分离株的胆汁应激反应。与目标1类似， 使用胆汁生长的阿克曼氏菌菌株和人上皮细胞的培养实验来测量 细菌结合效率和宿主免疫应答。本申请中提出的研究是 创新，因为它代表了一个实质性的脱离现状，提供了深入了解 生理多样性和整个阿克曼氏谱系的分子机制。拟议研究 是重要的，因为它将有助于定义整个谱系的生理景观，从而打开新的 促进或引入选择的阿克曼氏菌菌株的生物学信息治疗策略的视野 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.