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

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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.