Supplement: Microbially Generated Protons Mediate Interkingdom Host-Microbiome Cross-Talk and Maintain Intestinal Homeostasis Via Reciprocal Functions of Two Proton-Sensing GPCRs

Project Summary: Intestinal damage is observed in inflammatory bowel disease (IBD), enteric infections, as well as following surgical trauma, and environmental insults. Tissue restitution is a complex but coordinated cellular process to repair gut mucosa. We recently reported that the intestinal wound microenvironment specifically enriches a mucosa-­associated microbial consortium, which enhanced epithelial wound healing. The advances in next generation gene sequencing have identified diverse microbial species and metagenomes in the host;; however, to understand and exploit the potential of the microbiota’s >8 million genes and their metabolic functions, application of the mass-­spectrometry-­based identification of small metabolites and functional characterization by the methods of cell biology are crucial. Hence this proposed study will lay a foundation for my rigorous training in metabolome and cell biology of gut barrier function. For this purpose, we performed a preliminary analysis by mass spectrometry-­based global metabolomic profiling of intact and injured mucosa as well as colon of germ-­free and conventional mice. We found an elevated synthesis of microbial metabolites with low pKa, which are essential in establishing an extracellular pH gradient in the colonic crypts of the conventional mice, but not in germfree mice. We also found that the colonic epithelial cells of conventional mice express increased levels of proton-­sensing GPCRs (GPCR4 and GPCR68). Based on preliminary findings, the working hypotheses of this project are: 1) the injured mucosal tissue and its associated microbiota produce metabolites to regulate spatiotemporal changes in the proton concentration of the colon and thereby regulate intestinal homeostasis and repair, 2) microbiota-­produced metabolites modulate proton concentration, which is perceived by the proton-­sensing GPCRs of enterocytes to regulate gut permeability. Aim 1 is to confirm the role of microbiota-­produced metabolites in regulating cellular proliferation & repair via proton-­sensing GPCR4 and GPCR68 in the GPCR4-­/-­ and GPCR68-­/-­ mice, whose colonies are established in our animal facility. We will also perform the metabolomic profiling and RNA-­seq analysis of the colon of GPCR4-­/-­ and GPCR68-­/-­ mice. Furthermore, we will determine the metabolome of the injured mucosa in the germ-­free mouse. Specific Aim 2 will determine the function of the proton gradient & also the role of specific metabolites in the regulation of gut permeability by tight-­junction. University of Kentucky boasts an intellectually rich research environment whose resources will be used to carry out the proposed research as well as the mas-­spectrometry center at the Markey Cancer Center, University of Kentucky. The proposed research, in combination with a structured team of mentor, advisors, and collaborators as well as the training plan that includes didactic courses and workshops, is designed to facilitate my long-­term goal of developing an independently-­funded research program in chronic gastrointestinal inflammatory diseases, such as IBD, consistent with the mission of the NIDDK.