Deciphering the Pathogenesis of EHEC Infection and the Effects of Bacteria-Based Therapies Using Comparative Gut-on-a-Chip

项目摘要 这个NIH ORIP R21奖申请描述了一个为期2年的计划，旨在让我们调查 犬肠出血性大肠杆菌感染的病理生理学研究 和人类体外模型。肠出血性大肠杆菌胃肠道损伤的调查与研究进展 治疗干预的评估由于缺乏可翻译的体外模型而受到阻碍， 有效地再现了典型的人类结肠疾病，其发展为血性腹泻和溶血性腹泻， 尿毒症综合征（HUS）。狗感染肠出血性大肠杆菌的临床过程与人类非常相似， 犬肠出血性大肠杆菌感染可自然地导致轻度至重度形式的肠出血性大肠杆菌感染以及溶血尿毒综合征。我们搞 在拟议的研究中，犬和人类EHEC模型的开发将完成， 利用我们在兽医胃肠病学、肠道干细胞生物学、微生物组和微流体方面的专业知识， 器官芯片技术我们提出的研究是一个高风险的项目，因为犬EHEC体外模型 以前从未在器官芯片平台上进行过感染。然而，我们相信我们之前的工作 使得这项研究通过掺入类结肠素来开发犬和人EHEC芯片是高度可行的， 微生物组、免疫细胞、肠出血性大肠杆菌和基于细菌的治疗纳入生理模型。具体来说，Aim 1将允许开发犬和人类EHEC芯片，并评估微生物组在EHEC中的贡献。 疾病发病机制，同时利用单细胞水平的多组学（特别是 基因组学和转录组学）和RNA原位杂交。目标2.将允许评估免疫 在发病机制中的作用。目标3。将允许进一步评估的预防或治疗效果 两种充分研究的细菌（E. coliNissle 1917和产生MccPDI的E. coliNissle 1917）。大肠杆菌）。符合 ORIP的使命声明促进兽医科学家利用他们在比较医学方面的专业知识， 调查人类疾病，我的研究将使我能够利用我在比较胃肠病学方面的专业知识， 以及在原代干细胞培养中研究与肠出血性大肠杆菌感染相关的肠道内稳态的改变。 该建议中产生的结果对体内犬EHEC模型具有直接影响，并最终 人EHEC患者，因为他们将在实验设计中利用供体来源的类结肠， 新的见解，转录组学的改变，在启动，传播，或改善肠出血性大肠杆菌感染。我们的研究结果 在宿主反应中的物种相似性和差异可以应用于各种慢性疾病， 与人和狗中发生的肠道生态失调有关（即，结肠直肠癌， 糖尿病和阿尔茨海默病，仅举几例）。总之，本申请中提出的研究 将使我们能够绘制宿主-EHEC串扰以及基于细菌的治疗的效果， 单细胞多组学分析，这可能会对发现潜在疾病产生更广泛的影响 机制和开发新的预防性或治疗性抗EHEC疗法。

PROJECT SUMMARY This NIH ORIP R21 award application describes a 2-year plan designed to allow us to investigate pathophysiology of Enterohemorrhagic Escherichia coli (EHEC) infection in translational and comparative canine and human in vitro models. Investigation of the gastrointestinal injuries caused by EHEC and the development and assessment of therapeutic interventions have been hampered by the lack of translatable in vitro models that effectively reproduces the typical human colonic disease that progresses to bloody diarrhea and hemolytic uremic syndrome (HUS). The EHEC infection in dogs takes very similar clinical courses to that in humans as canine EHEC infection can naturally lead to mild to severe forms of EHEC infection as well as HUS. In carrying out the proposed research, development of both canine and human EHEC models will be accomplished while utilizing our expertise in veterinary gastroenterology, intestinal stem cell biology, microbiome, and microfluidic organ-on-chip technology. Our proposed study is a high-risk project because canine in vitro modeling of EHEC infection has never been performed before in an organ-chip platform. However, we believe our previous work makes this study highly feasible to develop canine and human EHEC Chips by incorporating colonoids, microbiome, immune cells, EHEC, and bacteria-based treatments into the physiological model. Specifically, Aim 1 will allow development of canine and human EHEC Chips and assessing the contribution of microbiome in the disease pathogenesis while mapping host responses by utilizing single-cell level multi-omics (especially genomics and transcriptomics) and RNA in situ hybridization. Aim 2. will allow assessment of immune contribution in the pathogenesis. Aim 3. will allow further assessment of preventative or therapeutic effects of two well studied bacteria (E.coli Nissle 1917 and MccPDI producing E. coli) on EHEC Chips. Consistent with the ORIP’s mission statements promoting veterinary scientists to employ their expertise in comparative medicine to investigate human diseases, my research will allow me to use my expertise in comparative gastroenterology as well as in primary stem cell culture to investigate alterations in intestinal homeostasis relevant to EHEC infection. The results generated in this proposal have direct implications for in vivo canine EHEC models and ultimately to human EHEC patients, since they will utilize donor-derived colonoids in the experimental designs and provide new insights into transcriptomic alterations in initiating, propagate, or ameliorate the EHEC infection. Our findings on the species similarities and differences in host responses can be applied to various chronic conditions that have been associated with intestinal dysbiosis that occur in both human and dogs (i.e., Colorectal Cancer, Diabetes Mellitus, and Alzheimer’s Disease, to name a few). In summary, the proposed study in this application will allow us to map the host-EHEC crosstalk as well as the effect of bacteria-based therapies by leveraging the single-cell multi-omics analysis, which may lead to a broader impact on uncovering the underlying disease mechanism and developing new preventative or therapeutic anti-EHEC therapy.