Novel high-throughput in vivo approach to define pathobionts driving colitis

摘要 炎症性肠病（IBD）患者经历慢性肠道炎症， 这是对微生物群不适当的免疫反应的结果。IBD患者， 尤其是克罗恩病（CD），发生合并症的风险更大，包括 肠纤维化/狭窄和IBD相关的结肠直肠癌（CRC）。这些疾病可以 由特定的大肠杆菌菌株驱动。CD和CRC微生物组含有高浓度的 大量的粘膜E.大肠杆菌被描述为“粘附侵袭性E. coli”（AIEC）。AIEC没有基因组 定义，而是通过通过详尽的体外试验测试的功能属性进行区分 共培养测定：粘附和侵入培养的上皮细胞并复制和 存在于巨噬细胞中。我们的初步数据表明，这种体外AIEC定义可能不 预测在体内粘膜定植，和特定的类群扩大与E。大肠杆菌，有助于 炎症性疾病。这项建议的目的是彻底审问AIEC 体内定义，定义由高水平E.杆菌 结肠和回肠组织，并确定候选因子窝藏的E。大肠杆菌， 发炎肠道的定植。为此，我们开发了一种多微生物 殖民策略，使用一种新的条形码技术， 临床E.大肠杆菌在一个复杂的社区。我们有 我使用一系列特征良好的临床AIEC和非AIEC来制定这一策略 分离自CD和健康患者肠粘膜的菌株。这种新方法 克服了与密切分析细菌宏基因组相关的技术限制 使用分子条形码细菌菌株与宿主组织相关联， 在我们的实验室中建立良好的模型，以及条形码靶向的高通量测序， 基因组学这种创新的方法使我们能够全面地询问AIEC 定义和鉴定发炎肠道定植所需的分子特征。我们 必须了解哪些微生物特征促进了高危大肠杆菌的粘膜定植。杆菌 菌株，以确定IBD患者在复杂的疾病过程的风险。

ABSTRACT Patients with Inflammatory Bowel Diseases (IBD) experience chronic intestinal inflammation as a consequence of inappropriate immune responses to the microbiota. Patients with IBD, particularly Crohn’s disease (CD), are at a greater risk of developing co-morbidities including intestinal fibrosis/stricturing and IBD-associated colorectal cancer (CRC). These diseases can be driven by specific strains of Escherichia coli. The CD and CRC microbiomes harbor high loads of mucosal E. coli described as “adherent-invasive E. coli” (AIEC). AIEC have no genomic definition, but instead are distinguished by functional attributes tested through exhaustive in vitro co-culture assays: the ability to adhere to and invade cultured epithelial cells and replicate and persist in macrophages. Our preliminary data indicate that this in vitro AIEC definition may not predict in vivo mucosal colonization, and that specific taxa expand with E. coli and contribute to inflammatory disease. The objectives of this proposal are to thoroughly interrogate the AIEC definition in vivo, define microbiome compositional changes driven by high levels of E. coli in colonic and ileal tissues, and identify candidate factors harbored by E. coli that permit colonization of the inflamed intestine. To this end, we have developed a polymicrobial colonization strategy that uses a novel barcoding technology to easily distinguish genetically similar, but functionally distinct sub-strains of clinical E. coli in a complex community. We have developed this strategy using a collection of well-characterized clinical AIEC and non-AIEC strains isolated from the intestinal mucosa of CD and healthy patients. This novel approach overcomes technological limitations associated with profiling bacterial metagenomes intimately associated with host tissues using molecular barcoded bacterial strains, gnotobiotic mouse models well-established in our lab, and barcode-targeted high-throughput sequencing and genomics. This innovative approach positions us to comprehensively interrogate the AIEC definition and identify molecular features required for colonization of the inflamed intestine. We must understand what microbial features promote mucosal colonization with high-risk E. coli strains in order to identify IBD patients at risk for a complicated disease course.