Title: Distal gut microbiome targets of host anti-proteolytic proteins during colitis ABSTRACT Our long-term goal is to advance our understanding of the distal gut microbiome from its basis in genomics with chemical biology and quantitative metaproteomics approaches. Over the last several years, we have developed a combination of chemical probe-based technologies, liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics, and bioinformatic analyses as approaches to interrogate and quantitate specific enzyme families in microbiome proteomes. We recently showed that colitic mice have a dramatic elevation in microbiome-secreted proteases within the distal gut relative to healthy mice using these combined methods. Correspondingly, we observe a significant increase in host anti-proteolytic proteins (APPs). Gut inflammation can be abated in colitis mouse models upon introduction of human APPs a-1-antitrypsin or elafin. While we posit that a-1-antitrypsin and elafin (and other APPs) reduce inflammation via inhibition of aberrant proteolytic activities, both APPs also have antimicrobial activity. Thus, the anti-inflammatory mechanism by which antitrypsin and elafin act could be primarily, or in part, attributable to altering bacterial composition. Our primary question is if APPs target microbial proteases. Here, we will elucidate the microbiome (and host) protein targets of six APPs overproduced in response to distal gut inflammation. These six APPs collectively target proteases from the serine, cysteine, and metallo subfamilies. We have purified all APPs of interest and aim to employ the proteins as ?bait? to determine if these APPs primarily target the inhibition of bacterial proteases. We will subsequently identify and quantitate the APP-captured proteins with our LC-MS/MS metaproteomics methods. We propose to focus our APP-enrichment strategy and metaproteomics on a well- established adoptive T cell transfer murine model of chronic colitis and directly compare to control mice raised under identical conditions. Our preliminary data confirms that a-1-antitrypsin irreversibly binds a unique collection of secreted proteins within colitic microbiome samples compared to controls. This is significant, as expression levels of host elastase and trypsin (targets of a-1-antitrypsin) are similar between the colitis and control groups. Over the next two years, we anticipate that we will identify several families of host APP-targeted bacterial proteases upregulated in colitic mice, as classified by sequence and/or structural homology. This list of proteases will help guide and focus future biochemical, cell-based, and animal model research on specific protease families as potential inflammatory agents (and therapeutic targets), as well as provide an optimized APP-enrichment methodology for translation into human IBD patients and other microbiome-related diseases.
The human intestinal tract is colonized with a vast and diverse network of commensal bacteria that are essential for human metabolism, immune development, and homeostasis, and protection from infiltration of pathogenic bacteria. Our recent studies found an excess of host anti-proteolytic proteins as well as microbial proteases are secreted into the gut lumen in a mouse model of colitis. The goal of this application is to identify all luminal proteins that bind to six overly abundant host anti-proteolytic proteins and determine if microbiome-derived secreted proteases are the primary targets.
