Interrogating the Cellular Mechanisms of Host-Pathogen Interactions during IBD

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DESCRIPTION (provided by applicant): Inflammatory bowel disease (IBD) is defined as severe, chronic inflammation of the intestinal mucosa, resulting in vomiting, diarrhea, bleeding, abdominal pain and weight loss. Current estimates suggest that 3-to-4 million people suffer from IBD and more are diagnosed each year. The cause(s) of IBD is not known and likely to be multi-factorial. Many lines of evidence suggest that the initiation of IBD involves abnormal host-microbial interactions at the intestinal epithelium. One microorganism that is commonly associated with the intestinal mucosa of IBD patients but not from healthy subjects is called adherent-invasive E. coli (AIEC). AIEC are a new pathotype defined by their ability to adhere to and invade epithelial cells in the absence of known virulence factors associated with other pathogenic E. coli. A comprehensive analysis of the cellular response to AIEC infection has not been performed, and it is not clear whether the IBD gut environment selects for AIEC infection in humans. The overall goals of this grant are to A) define and compare how the proteome is altered in human epithelial cells infected with AIEC (Aim 1) and affected with IBD (Aim 2), and B) determine whether any of the observed differences in protein abundance from either condition (Aim 1&2) causes epithelial cells to become more permissive to AIEC infection. In vitro, cells infected with AIEC, but not other pathotypes or commensal E. coli, have increased levels of a protein marker of the autophagy pathway. Autophagy is a cell-intrinsic response that degrades cellular contents, which can be activated in response to intracellular infection with bacteria. Polymorphisms in autophagy genes in humans are also associated with a significantly higher risk of IBD. Therefore, I propose that altered levels of proteins in the autophagy pathway occur in human epithelial cells affected with IBD and infected with AIEC, leading to aberrant control of intracellular infection. To investigate this hypothesis, I will combine an unbiased proteomic approach using quantitative mass spectrometry with a complementary gain-of-function and loss-of-function approach to assess whether changes in the abundance of specific host proteins are necessary and/or sufficient to affect AIEC attachment, invasion, or intracellular persistence. These studies will identify AIEC- and IBD-induced changes to host proteins on a scale never previously possible and will elucidate pathways that are involved in the response to AIEC and in the induction of IBD. Our studies to characterize the host cell-intrinsic mechanism(s) that restrict AIEC infection may also reveal novel cellular drug targets that may be useful in developing "host- based" antibiotics capable of inhibiting intracellular infection, and/or diminishing the microbial component of IBD pathogenesis. By identifying cellular changes involved in the onset of IBD in patients, our proposal may also lead to the identification of IBD biomarkers to facilitate early diagnosis, a goal for interrupting the chronic cycle of IBD pathogenesis.