Intestinal damage is observed in inflammatory bowel disease (IBD), enteric infections,aswellasfollowingsurgicaltrauma,andenvironmentalinsults.Tissuerestitutionisacomplexbut 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 woundhealing.Theadvancesinnextgenerationgenesequencinghaveidentifieddiversemicrobialspeciesand metagenomesinthehost;?however,tounderstandandexploitthepotentialofthemicrobiota?s>8milliongenes andtheirmetabolicfunctions,applicationofthemass-spectrometry-basedidentificationofsmallmetabolitesand functional characterization by the methods of cell biology are crucial. Hence this proposed study will lay a foundationformyrigoroustraininginmetabolomeandcellbiologyofgutbarrierfunction.Forthispurpose,we performedapreliminaryanalysisbymassspectrometry-basedglobalmetabolomicprofilingofintactandinjured mucosa as well as colon of germ-free and conventional mice. We found an elevated synthesis of microbial metaboliteswithlowpKa,whichareessentialinestablishinganextracellularpHgradientinthecoloniccryptsof theconventionalmice,butnotingermfreemice.Wealsofoundthatthecolonicepithelialcellsofconventional miceexpressincreasedlevelsofproton-sensingGPCRs(GPCR4andGPCR68).Basedonpreliminaryfindings, theworkinghypothesesofthisprojectare:1)theinjuredmucosaltissueanditsassociatedmicrobiotaproduce metabolitestoregulatespatiotemporalchangesintheprotonconcentrationofthecolonandtherebyregulate intestinalhomeostasisandrepair,2)microbiota-producedmetabolitesmodulateprotonconcentration,whichis perceivedbytheproton-sensingGPCRsofenterocytestoregulategutpermeability.
Aim1 istoconfirmtherole ofmicrobiota-producedmetabolitesinregulatingcellularproliferation&repairviaproton-sensingGPCR4and GPCR68intheGPCR4-/-andGPCR68-/-mice,whosecoloniesareestablishedinouranimalfacility.Wewillalso perform the metabolomic profiling and RNA-seq analysis of the colon of GPCR4-/- and GPCR68-/- mice. Furthermore,wewilldeterminethemetabolomeoftheinjuredmucosainthegerm-freemouse.
SpecificAim2 willdeterminethefunctionoftheprotongradient&alsotheroleofspecificmetabolitesintheregulationofgut permeability by tight-junction. Emory University boasts an intellectually rich research environment whose resourceswillbeusedtocarryouttheproposedresearchaswellasthemas-spectrometrycenterlocatedacross thetowninGeorgiaInstituteofTechnology,Atlanta.Theproposedresearch,incombinationwithastructured team of mentor, advisors, and collaborators as well as the training plan that includes didactic courses and workshops,isdesignedtofacilitatemylong-termgoalofdevelopinganindependently-fundedresearchprogram inchronicgastrointestinalinflammatorydiseases,suchasIBD,consistentwiththemissionoftheNIDDK.
Importance of extracellular proton concentration is critical in intestinal mucosa during health and disease. However,thereisnoevidence-basedknowledgehowextracellularpHofthemucosalmicroenvironmentregulate intestinalrepairandrestorationofhomeostasis.Leveragingthemetabolomicstudies,thisprojectisdesignedto investigate two proton-sensing G-protein coupled receptors and their role in microbial metabolite-driven extracellularpHgradientduringmucosalhomeostasisandrepair.
