2:00 pm - 3:00 pm
410C Agriculture/Forestry Centre, Agriculture/Forestry Centre, Edmonton
A graduate exam seminar is a presentation of the student’s final research project for their degree.
This is an ALES MSc Final Exam Seminar by Stephanie Monteith. This seminar is open to the general public to attend.
Thesis Topic: Engineering agarolytic enzymes into human gut symbiont, Bacteroides thetaiotaomicron for the development of drug delivery systems to treat dysbiosis
MSc with Drs. Richard Uwiera, Trina Uwiera, and D. Wade Abbott.
The microbiota of the distal gastrointestinal tract of monogastric animals plays vital roles in maintaining host health; one of these roles includes the production of energy and nutrients from dietary polysaccharides that are indigestible by enzymes contained within the human genome. Dietary polysaccharides (prebiotics) foster the proliferation of beneficial bacteria within the microbiota by improved carbohydrate metabolism and the maintenance of stable microbial community structures within the distal gastrointestinal tract in order to achieve improved intestinal health. Furthermore, the co-administration of prebiotics with probiotics, or “synbiotics”, could augment the effects of both probiotics and prebiotics possibly further enhancing intestinal health. Agarose is a marine polysaccharide that is well suited to act as a selective prebiotic nutrient in the development of a designer synbiotic delivery system, as this unique polysaccharide is resistant to digestion by many intestinal microorganisms present within the gastrointestinal tract of terrestrial animals. Agarose can be completely saccharified into individual monosaccharide substituents by the combined enzymatic activities of three glycoside hydrolases (agarases): GH16B, GH117B, and GH2C. These agarases have been identified within the polysaccharide utilization locus of the human intestinal bacterium Bacteroides uniformis NP1. From a bioengineering perspective, it may be possible to incorporate the agarases expressed in Bacteroides uniformis NP1 into other intestinal bacteria for modifying agarose substrates within the intestine. This research project was first directed at engineering Bacteroides thetaiotaomicron (B. theta) a non-agarolytic gut bacterium, to produce agarase enzymes that hydrolyse agarose glycosidic linkages. The second area of my research focused on developing an assay to measure release of cargo from algal-polysaccharide-derived capsules. In the first objective, each gene product was engineered to express N-terminal signal peptides in an attempt to traffic proteins to the outer surface of the bacterium to provide the enzymes access to agarose substrates present in the extracellular medium. In my research project, the agarase genes were successfully introduced into the B. theta genome through homologous recombination, and all three agarases were produced by the bacterium in detectable amounts on western blots. Notably, enzymatic products were observed using thin layer chromatography following incubation with the engineered B. theta strains indicating the engineered strains’ abilities to hydrolyse agarose substrates. Agarose digestion assays using whole cell engineered B. theta strains showed activity consistent with the expression of the GH16B endo-β-agarase, indicating that this was the only enzyme expressed to the outer surfaces of the bacteria. Exogenous supplementation of the exo-agarases, GH117B and GH2C, to the medium confirmed this observation as bacterial growth was detected on agarose for strains expressing GH16B. From these observations, engineered B. theta strains expressing the GH16B agarase are capable of adequately hydrolysing agarose into smaller oligosaccharides. This suggests that an engineered strain of B. theta expressing GH16B agarase is technically possible, and may be the initial step in developing Bacteroides spp. bacteria as a component of an agarose- dependent synbiotic delivery system. This in turn, could enable the targeted release of bioactive molecules from capsules comprised of agar containing substrates for the treatment of enteric inflammatory disease and intestinal infections. To improve the efficacy of agarose hydrolysis, and as such, enhance the potential application of B. theta as a probiotic for the treatment of intestinal diseases, further research will be required to optimize the trafficking the agarases to the outer surface of the cell. This could include modifying signal peptides expressed on the GH117B and GH2C agarases to help deliver these enzymes to the outer membrane of the bacteria, or adding genes to express an oligosaccharide transporter and polysaccharide binding protein, namely B. uniformis NP1 AgPUL SusC-like and SusD-like proteins. The second objective was completed by measuring the oxidation activity of released HRP from porphyran- and carrageenan-derived capsules. The capsules were leaky and optimization of the integrity of the capsules as well as the cargo used would be important to confirm the polysaccharides efficacy of capsules to release therapeutic molecules within the distal GIT. Importantly, oligosaccharides observed from capsules digested with purified GH16 enzymes as well as GH16 enzymes produced from both unmodified and engineered bacteria support the development of a drug delivery system using algal polysaccharide derived capsules and engineered agarolysis bacteria to deliver therapeutic molecules within the distal GIT.