Genetic Dissection of Nutrient Acquisition in Diverse Akkermansia Strains

肠道微生物群与人类健康之间的联系已经建立，但许多居民仍然缺乏特征。嗜粘蛋白阿克曼氏菌是一种有益的肠道微生物，与宿主的代谢和免疫健康呈正相关。阿克曼氏菌通常占肠道微生物群的1- 4%，在某些个体中，它可以超过20%。尽管阿克曼菌普遍存在，但大多数阿克曼菌研究都集中在单一菌株上，影响定植的因素尚不清楚。 阿克曼氏菌是疣微菌门中唯一一种在哺乳动物肠道内定植的菌，它占据了一个独特的生态位，以宿主粘蛋白为食。我和其他人的工作表明，阿克曼氏菌表现出惊人的遗传多样性水平，至少有四个不同的类群，其中大部分仍然没有特征。值得注意的是，许多阿克曼氏菌缺乏同化硫酸盐还原的途径，这使得它们依赖于外源硫化物来产生含硫分子，如半胱氨酸。这种对减少硫的需求可以影响微生物-微生物相互作用，并有可能改变肠上皮细胞中的H2S水平。由于H2S作为一种具有强效抗炎作用的信号分子，局部H2S浓度的改变可能对宿主和周围微生物群的生态产生广泛的影响。 这项研究计划的长期愿景是使用遗传方法来揭示人类肠道微生物群成员中具有应变水平分辨率的殖民化的基本方面。根据这一愿景，本提案中概述的短期目标将揭示阿克曼氏菌生物学的基本特征和影响定植的菌株特异性相互作用。我们将调查：（1）阿克曼氏菌皮利与宿主糖蛋白之间的相互作用;（2）不同阿克曼氏菌菌株之间碳水化合物利用的遗传机制;（3）特定阿克曼氏菌菌株与产生H2S的肠道微生物之间的微生物-微生物相互作用。 由于我们独特的资源，我们的实验室处于追求这些目标的绝佳位置。这些包括阿克曼氏菌菌株的集合，阿克曼氏菌转座子突变体的库，以及在我的博士后工作期间开发的操纵阿克曼氏菌的遗传方法的知识，这在以前被认为是遗传上难以解决的。拟议的研究将提供微生物学，分子生物学，显微镜，高通量遗传筛选，生物信息学和动物模型的培训。 拟议的工作将有助于了解肠道微生物群的潜在关键成员的生态学。更广泛地说，这项研究对于更好地了解肠道微生物组复杂生态系统中基本的宿主-微生物和微生物-微生物相互作用具有重要意义。

The link between the intestinal microbiota and human health is well established, yet many of its inhabitants remain poorly characterized. Akkermansia muciniphila is a beneficial intestinal microbe that is positively correlated with the metabolic and immunologic health of its host. Akkermansia typically represents 1- 4% of the intestinal microbiota, and in some individuals, it can exceed 20%. Despite its prevalence, most Akkermansia research has focused on a single strain, and the factors that influence colonization are unknown.      Akkermansia is the only member of the Phylum Verrucomicrobia known to colonize the mammalian intestine, and it occupies a distinct niche where it forages on host mucins. Work from myself and others indicates that Akkermansia exhibits a striking level of genetic diversity with at least four distinct phylogroups, most of which remain uncharacterized. Notably, many Akkermansia lack pathways for assimilatory sulfate reduction, which renders them dependent on exogenous sulfide to produce sulfur containing molecules such as cysteine. This requirement for reduced sulfur can influence microbe-microbe interactions and has the potential to alter H2S levels at the intestinal epithelium. Because H2S acts as a signalling molecule with potent anti-inflammatory effects, alterations of local H2S concentrations could have broad implications for the host and the ecology of the surrounding microbiota.      The long-term vision for this research program is to use genetic approaches to uncover fundamental aspects of colonization among members of the human intestinal microbiota with strain level resolution. In line with this vision, the short-term objectives outlined in this proposal will reveal basic features of Akkermansia biology and the strain-specific interactions influencing colonization. We will investigate: (1) interactions between Akkermansia pili and host glycoproteins; (2) the genetic mechanisms of carbohydrate utilization among diverse Akkermansia strains; and (3) microbe-microbe interactions between specific Akkermansia strains and H2S producing intestinal microbes.      Our lab is in an excellent position to pursue these aims because of our unique resources. These include a collection of Akkermansia strains, a library of Akkermansia transposon mutants, and knowledge of genetic approaches developed during my postdoctoral work to manipulate Akkermansia, which was previously considered genetically intractable. The proposed research will provide training in microbiology, molecular biology, microscopy, high throughput genetic screens, bioinformatics, and working with animal models.      The proposed work will contribute to understanding the ecology of a potential keystone member of the intestinal microbiota. More broadly, this research has important implications for better understanding basic host-microbe and microbe-microbe interactions in the complex ecosystem of the intestinal microbiome.