LEAPS-MPS: Exploring the Role of Urinary Host N-Glycans as a Nutrient Source for Bladder Bacteria Metabolism

In this project, funded by the MPS-LEAPS (Launching Early-Career Academic Pathways) Program and managed by the Division of Chemistry (CHE), Professor Jean-Philippe Gourdine and his students at Lewis & Clark College will perform studies focused on the development of biochemical methods to identify commensal bladder bacteria capable of symbiotic nutrient acquisition of host N-glycans and to elucidate the molecular mechanisms of glycoside hydrolases (GHs) involved in this process. Urine is a hostile environment for bacterial growth; nevertheless, low-biomass microorganisms (urobiome) can live in the bladder lumen. What do these tiny tenants survive on? Previous bioinformatics work has shown that several bacterial members of the urobiome can digest host glycoproteins. Uromodulin (UMOD) is the most concentrated N-glycoprotein in urine. Could UMOD be a nutrient source for the urobiome? Professor Gourdine and his students will address this question by characterizing urinary bacterial interactions with UMOD and by cloning, expressing, and functionally characterizing putative bacterial GHs involved in N-glycans degradation. Their studies could provide valuable knowledge to model the urobiome ecology toward host N-glycans. This research, conducted with paid-research students and in a course-based undergraduate research experience (CURE), will integrate outreach activities with the biotechnology industry in Portland, Oregon.

The biochemical integrity of purified UMOD will be assessed by SDS-PAGE, immunoblot, lectin-blot, and mass spectrometry. Urinary bacteria will be grown either in liquid media or solid media enriched with purified UMOD. For liquid culture, UMOD digestion will be assessed biochemically as described above, and bacterial growth on UMOD will be monitored time-dependently. To identify UMOD binders and consumers, UMOD covalently bound to magnetic beads will be incubated with urinary bacterial pellets or selected bladder bacteria. Taxonomic identification of UMOD binders or consumers will be determined by 16S rRNA metagenomics. In a CURE setting, bacterial GHs involved in N-glycans degradation will be cloned into pET vectors, expressed, and purified by Immobilized Metal Affinity Chromatography. GHs’ purity and sizes will be verified by SDS-PAGE and anti-His tag immunoblot. Enzymatic characterizations will be performed by SDS-PAGE, HPLC, and mass spectrometry using purified glycosylated UMOD. Kinetic parameters will be determined by fluorescent enzymatic assays with appropriate fluorescent substrate (e.g., 4-methylumbelliferyl- ⍶ -D-mannopyranoside for GH38), and isothermal microcalorimetry. Further 3D structural characterization will be carried out by X-ray protein crystallography.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Award Number: 2532889
Principal Investigator: Jean-Philippe Gourdine
Funds Obligated: $249,263
State: OR