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Mslati, Matthias Ahmad

University of British Columbia

Background The nutritional program(s) used by enteric bacterial pathogens to colonize their host’s intestines remain unclear. To begin to understand this question, we focused on the enteric murine pathogen Citrobacter rodentium. C. rodentium has to cross a thick mucus barrier, which is primarily made up of Muc2 glycoprotein, during its infection of the large intestine. As a glycoprotein, Muc2-linked sugars can constitute a potential nutrient source for C. rodentium. This thesis examined the nutritional role and dynamic changes of Muc2-linked sugars during C. rodentium infection. Methods In vitro growth assays were used to assess whether C. rodentium can utilize the Muc2-linked sugars: N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc), galactose (Gal), and fucose (Fuc). Stool samples of mice at baseline and during different stages of C. rodentium infection were analyzed for the sugar concentration using an advanced quantitative technique termed UHPLC/QqQ-MS. Lastly, mutant strains of C. rodentium lacking key genes for the metabolism of Muc2-linked sugars were generated and assessed for their fitness to colonize and cause pathological damage to mice. Results We found that C. rodentium was able to grow on the Muc2-linked sugars: GlcNAc, NeuNAc, and Gal. We quantified the levels of these 3 sugars in the stool samples of mice, and determined that they are primarily Muc2 derived and that GlcNAc levels are increased during C. rodentium infection. In vivo competition experiments revealed that C. rodentium mutant strains with defects in two or more of the Muc2-linked sugar pathways were outcompeted by the wild type (WT) strain. Lastly, a C. rodentium mutant strain unable to use all 3 sugars (Δ3) displayed a significant delay in colonizing mice and reaching the intestinal epithelium. As a result, the majority of mice infected with this mutant displayed no pathological damage to their colons, indicating a major defect in virulence. Conclusions We show that the intestinal Muc2-linked sugars serve as critical nutrient sources for C. rodentium during colonization of the mouse intestine, and that the levels of Muc2-linked sugars are influenced by C. rodentium infection suggesting a potential role of C. rodentium in shaping the nutrient environment in the intestine.

Text

2023-07-04

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/85187

Experimental Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/