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

摘要 炎症性肠病(IBD)患者的慢性肠道炎症表现为 这是对微生物区系不适当的免疫反应的结果。IBD患者， 尤其是克罗恩病(CD)，患上共病的风险更大，包括 肠纤维化/狭窄和IBD相关性结直肠癌(CRC)。这些疾病可能 是由特定的大肠杆菌菌株驱动的。CD和CRC微生物群含量高 粘附性侵袭性大肠杆菌(AIEC)。AIEC没有基因组 定义，但取而代之的是通过体外详尽测试的功能属性来区分 共培养实验：黏附和侵袭培养的上皮细胞并复制和 持续存在于巨噬细胞中。我们的初步数据表明，这种体外AIEC定义可能不会 预测在体内的粘膜定植，以及特定的分类群随着大肠杆菌的扩张而有助于 炎症性疾病。这项提议的目的是彻底审问AIEC 定义体内，定义由高水平的大肠杆菌在体内驱动的微生物组组成的变化 结肠和回肠组织，并确定大肠杆菌所含的候选因子 炎症性肠炎的定植。为此，我们开发了一种多菌种 一种殖民策略，使用一种新的条形码技术来轻松区分基因 在一个复杂的社区中，临床大肠杆菌的亚株相似，但功能不同。我们有 使用一组具有良好特征的临床AIEC和非AIEC制定了这一策略 从CD患者和健康患者的肠粘膜分离到的菌株。这一新方法 克服了与密切分析细菌元基因组相关的技术限制 利用分子条形码细菌菌株与宿主组织相关联的灵生小鼠 我们实验室中成熟的模型，以及针对条形码的高通量测序和 基因组学。这种创新的方法使我们能够全面审问AIEC 定义和鉴定炎症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 以确定具有复杂病程风险的IBD患者。

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.