University of Lethbridge Theses

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Butyrate, Bacteroides thetaiotaomicron and Campylobacter jejuni modulate the expression of beta-defensins, Toll-like receptors and cytokines in Caco-2 cells
(Lethbridge, Alta : University of Lethbridge, Dept. of Chemistry and Biochemistry, 2016) Yamashita, Justin; University of Lethbridge. Faculty of Arts and Science; Abbott, D. Wade; Mosimann, Steven C.
Dietary fibre fermentation produces short-chain fatty acids, including butyrate (NaB). NaB increases histone tail acetylation within the chromatin of colonic cells, a process associated with increased gene expression. Campylobacter jejuni a prominent foodborne pathogen triggers inflammatory enteritis, whereas NaB and the commensal enteric bacterium Bacteroides thetaiotaomicron (B. theta) have documented anti-inflammatory properties. In this study I have developed a model system for studying the NaB-colonocyte-bacteria interaction and have used it to investigate responses to NaB, C. jejuni and B. theta using an in-house immunomodulatory gene array. NaB induced significant levels of transcription for nearly every immunomodulatory gene. Further, NaB and B. theta stimulate the expression of defensins and Toll-like receptors, while C. jejuni decreased both. This study provides mechanistic insights into the human colonocyte immune response and presents several gene targets as potential biomarkers for inflammatory expression responses in vivo.
Characterization of NfGHX : a founding member of a new glycoside hydrolase family from the anaerobic fungus Neocallimastix frontalis
(Lethbridge, Alta : University of Lethbridge, Dept. of Biological Sciences, 2016) Uddin, Muhammed Salah; University of Lethbridge. Faculty of Arts and Science; Abbott, D. Wade; Selinger, L. Brent
The discovery and characterization of enzymes that catalyze new reactions, and increase the rate or efficiency of bioconversion are promising lines of biotechnological research. In this regard the rumen anaerobic fungi represent an underexploited source of novel carbohydrate active enzymes that are active on recalcitrant plant cell wall polysaccharides. In this study, four rumen fungal genes (Nf2152, Nf2215, Nf2523 and Pr2455) were identified as functional CAZymes that represent founding members of a new GH family, referred to here as ‘GHX’. Two members of GHX, Nf2152 and Nf2523, were functionally characterized, and it was discovered that they have two different activities. Nf2152 releases a single product (β-1,2-arabinobiose) from sugar beet arabinan (SBA), and β-1,2-arabinobiose and a galactoarabinose with an unknown linkage from rye arabinoxylan (RAX). Nf2523 exclusively produces galactoarabinose from RAX. To the best of my knowledge, this represents the first reported galactoarabinobiosidase. Both β-1,2-arabinobiose and galactoarabinose are not conventional structures within SBA and RAX, and in fact are more likely released from the glycan side chains of arabinogalactan proteins (AGPs) that co-purify with the polysaccharides. In this regard, the GHXs studied here represent valuable tools for sequencing diverse AGP glycans and assisting in their bioconversion for industrial applications, such as animal production.
Direct visualization of yeast mannan metabolism in bovine-adapted Bacteroides thetaiotaomicron strains at the single cell level using fluoresceinamine - yeast mannan conjugates
(Lethbridge, Alta. : Universtiy of Lethbridge, Department of Biological Sciences, 2019) Klassen, Leeann; University of Lethbridge. Faculty of Arts and Science; Abbott, D. Wade; Yevtushenko, Dmytro P.
The rumen of beef cattle houses a diverse community of microorganisms that impact feed digestion, nutrient accessibility, host health, and waste production. Fibrolytic enzymes, probiotics, and prebiotics are promising candidates for next-generation feed additives, with the goal of inducing beneficial changes to the rumen microbiome. Current methods to investigate the mechanistic interactions between bacteria and feed glycans are “indirect” and lack the required sensitivity to validate probiotic-prebiotic application. To address these limitations, I have extended fluorescent glycan conjugates (FGCs) to visualize polysaccharide uptake in bacterial isolates and complex ecosystems at the single-cell level. Yeast α-mannan (YM) was conjugated to 6-aminofluorescein and fed to pure cultures of Bacteroides thetaiotaomicron VPI-5482, a well-studied intestinal symbiont that metabolizes YM, and closely related bovine-adapted bacterial isolates. Uptake of FGCs, coupled to complementary genomic and transcriptomic analysis, provided direct evidence of individual genotypes endowed with YM metabolic potential in pure and complex culture.
Engineering dual-glycan responsive expression systems for tunable production of heterologous proteins in Bacteroides thetaiotaomicron
(Lethbridge, Alta. : Universtiy of Lethbridge, Department of Biological Sciences, 2018) Smith, Marshall B.; University of Lethbridge. Faculty of Arts and Science; Abbott, D. Wade; Selinger, L. Brent
Genetically engineering symbiotic bacteria remains an underexploited opportunity to improve host-health and create new classes of biological devices, such as diagnostics or intestinal delivery systems for therapeutics. Bacteroides thetaiotamicron (B. theta) is a Gram-negative intestinal anaerobe with potential for the capability to produce functional heterologous proteins within a host intestine. To improve the strength and regulatory fidelity of transgene expression in B. theta, I have developed platform expression strains with engineered regulatory proteins under control of promoter elements that respond to dextran and arabinogalactan, two chemically distinct glycans. In addition to single glycan induction, I have also developed a novel “dual-glycan” expression system that requires the addition of both dextran and arabinogalactan for induction. Additionally my engineered strains are compatible with a series of chromosomal integration and episomal vectors that improve the throughput of gene cloning, integration, and expression. Together this expression system provides a new collection of glycan-responsive tools to improve transgene expression in B. theta and provides the proof-of-concept for engineering more complex dual-glycan expression systems.
Glycomic analysis of the in vitro modification and utilization of chicken intestinal mucin O-glycan carbohydrates by the enteric poultry pathogen Clostridium perfringens
(Lethbridge, Alta. : Universtiy of Lethbridge, Department of Biological Sciences, 2019) MacMillan, Jaclyn L.; University of Lethbridge. Faculty of Arts and Science; Abbott, D. Wade; Selinger, L. Brent
Clostridium perfringens is the etiological agent of necrotic enteritis (NE) in poultry. NE occurs within the small intestine and is characterized by intestinal mucus depletion and tissue inflammation. This disease costs the global poultry industry an estimated USD $2-6 billion per year. The small intestine mucus glycans of birds contain a large abundance of sialic acid (Neu5Ac) and sulfate, which may impede colonization by enteric avian bacterial pathogens. C. perfringens was shown here to liberate and metabolize Neu5Ac from extracted broiler chicken mucin O-glycans. By correlating these findings with carbohydrate active enzymes active on mucin O-glycans and the core metabolic enzymes present within C. perfringens, a biochemical basis for these relationships can be established. These new findings suggest that Neu5Ac utilization is significant for mucus colonization by C. perfringens and may provide C. perfringens with a competitive advantage in the avian intestine
Heterologous expression and secretion of nanobodies targeting Campylobacter jejuni for intestinal health applications
(Lethbridge, Alta : University of Lethbridge, Dept. of Chemistry and Biochemistry, 2016) McLean, Richard; University of Lethbridge. Faculty of Arts and Science; Abbott, D. Wade; Mosimann, Steven C.
As strategies for engineering the enteric microflora continue to advance, the vision of a future with a secondary, artificial immune system increasingly comes into focus. There are numerous challenges that will need to be overcome before this is a reality including the construction of a chassis capable of producing functional antimicrobial compounds at adequate concentrations and the construction of libraries of antimicrobial compounds capable of targeting a range of pathogens including those that develop resistance. Towards these ends, I have elected to engineer Bacteroides thetaiotaomicron (B. theta), one of the most prevalent and stable organisms in the human distal gut, to heterologously express and secrete nanobodies that bind the flagella of Campylobacter jejuni. Nanobody genes were inserted behind native B. theta promoters and integrated into the genome allowing for induction following the introduction of a specific inducing compound. Signalling peptides were fused to the nanobodies allowing for targeting the nanobodies out of Escherichia coli (E. coli) cells. Also as part of this project, novel signal peptides have also been characterized allowing for the targeting of protein to any subcellular compartment within a gram-negative bacterium.
Investigation of the structure and digestibility of fluorescently labeled carbohydrates using glycoside hydrolases
(Lethbridge, Alta : University of Lethbridge, Dept. of Biological Sciences, 2016) Anele, Anuoluwapo; University of Lethbridge. Faculty of Arts and Science; Abbott, D. Wade; Selinger, L. Brent
Developing alternatives to the use of antimicrobials is essential to maintaining the sustainability of animal production and lowering the prevalence of antimicrobial resistance. Prebiotics have not been readily adopted due to limited knowledge of the mechanisms driving prebiotic-bacteria-host interactions. Presented here are preliminary insights into the utility of three prebiotic compounds: neoagarooligosaccharides (NAOS) mannooligosaccharides (MOS), and pectic oligosaccharides (POS). The activity of three agarases from Bacteroides uniformis NP1: BuGH86 (an endolytic enzyme that produces NAOS from agarose), as well as BuGH2C and BuGH117B (exolytic enzymes that remove 3,6-anhydro-L-galactose from the non-reducing end of NAOS and D-galactose from the non-reducing end of agarooligosaccharides, respectively) were determined. NAOS, MOS and POS were then used as substrates to generate fluorescent probes for evaluating interactions with bacterial enzymes. For NAOS and MOS, the digestion of ANTS-labeled oligosaccharides was similar to that of native versions; however, digestion of ANTS-labeled POS was considerably hindered.
Production and secretion of heterologous immunomodulators in bacteroides thetaiotaomicron
(Lethbridge, Alta. : University of Lethbridge, Dept. of Biological Sciences, 2019) Tingley, Jeffrey; University of Lethbridge. Faculty of Arts and Science; Selinger, L. Brent; Abbott, D. Wade
Chronic and acute inflammation in the colon significantly impact human and animal health, and productivity in agriculture. Unfortunately, current treatments are not very effective for long-term management and treatment is costly. This research project has helped address these limitations by engineering a colonic bacterium, Bacteroides thetaiotaomicron, to produce tumor necrosis factor receptor 2 (TNFR2), α-1-antitrypsin (A1AT) and elafin. These anti-inflammatory proteins were stitched to BtAnchor and BvSecrete signal peptides that facilitate their trafficking to the outer surface and external environment of the cell, respectively. Protein targets were initially produced and localized within the Gram-negative, model organism, Escherichia coli, and elafin demonstrated statistically significant inhibition of elastase. Protein targets: A1AT, TNFR2, and elafin were successfully produced in a dual-glycan responsive B. theta strain previously developed in the Abbott lab. The strains developed as part of this project represent novel tools for reducing of inflammation in agricultural livestock and human medicine.
Understanding the recognition and utilization of homogalacturonan by Bacteroides thetaiotaomicron
(Lethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry, 2014) Farnell, Benjamin J.; Abbott, D. Wade; Mosimann, Steven C.
Homogalacturonan (HG) is a structural plant cell wall polysaccharide and a key source of dietary fiber. The human genome does not contain a single enzyme known to be involved in pectin digestion, and therefore, in order to modify HG fibers and potentially extract nutritional value, humans rely on a consortium of symbiotic intestinal bacteria, such as Bacteroides thetaiotaomicron, to deconstruct and to ferment this complex carbohydrate into host absorbable products. B. thetaiotaomicron contains over 300 predicted carbohydrate active enzymes within its genome that are primarily organized into sugar-selective metabolic pathways called Polysaccharide Utilization Loci PULs (PULs). One such PUL (PUL75: BT4108-BT4124), is activated by HG and is believed to contain enzymes that convert polymerized HG into monosaccharides (GalA). This study reports molecular biology, biochemistry, and functional genomics data that characterize the function of PUL75 gene products involved in HG utilization. Based on these findings, a model for the step-wise process of HG recognition, transport and modification by PUL75 is proposed.