Microbiota based mechanisms of post-infection irritable bowel syndrome

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ABSTRACT One in six adults in the U.S. suffers from chronic and often disabling symptoms of irritable bowel syndrome (IBS). Intestinal infections are an established risk-factor for development of post-infection IBS (PI-IBS). The intestinal tract contains a variety of proteases, and we have discovered that PI-IBS patients have significantly higher fecal proteolytic activity (PA) than controls. More importantly, PA associates strongly with loss of intestinal barrier function and worse symptoms for the patients. We found that patients who develop PI-IBS and have high PA have a significant loss of microbial diversity that starts soon after the infection. Key microbial taxa are lost, especially from the Alistipes genus. Using metaproteomics, we found that proteases driving PA in these patients are of human origin. In order to understand if loss of microbiota could be affecting host proteases, we used germ-free mice. Colonization of germ-free mice with healthy human microbiota (humanization) results in a significant decline of PA suggesting commensal microbes inhibit host proteases and thus have a role in maintaining intestinal health. However, the dysbiotic microbiota from the high PA patients that are missing specific microbes were unable to suppress PA. We hypothesize that Alistipes and other missing bacteria play a critical role in suppression of PA. We plan to test the candidate bacteria identified in the preliminary experiments and inter-species interactions in PA regulation in Aim 1. Next, we determined how loss of microbes result in poor inhibition of proteases. Unconjugated bilirubin is an inhibitor of serine proteases and microbial β-glucuronidases deconjugate bilirubin. We found that the PI-IBS patients with high PA have lower fecal microbial β-glucuronidase enzymatic activity. Additionally, they have lower levels of end products of bilirubin deconjugation. β-glucuronidases are a large family of microbial enzymes with varying sources, structures and catalytic efficacies for different substrates. We hypothesize that loss of specific microbial β-glucuronidases will result in impaired deconjugation of bilirubin. In Aim 2, we will analyze metagenomics data from our PI-IBS patients with high and low PA for presence of microbial β-glucuronidases as well as determine the efficacy of these fecal samples for bilirubin deconjugation. Additionally, we will generate purified β-glucuronidases from Alistipes and other bacterial taxa for assessing bilirubin deconjugation efficacy in vitro. Next, we have shown that fecal microbiota transfer using an Alistipes enriched low PA community can suppress PA in high PA humanized mice providing a rationale for using microbiota for protease suppression and correcting intestinal barrier function. In Aim 3, we will use cohousing strategies to allow microbiota transfer between humanized high and low PA mice and determine if barrier dysfunction associated with a high PA state can be reversed. Furthermore, we will determine changes in barrier pathways, ionic selectivity and expression of tight junction proteins upon engraftment of new microbiota. Together, these aims will examine microbial influence on PI-IBS pathophysiology via regulation of intestinal proteases. Identification of microbiota-based strategies that can result in protease inhibition and restoration of barrier function can be beneficial for IBS and other conditions associated with microbial dysbiosis.