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Title:Polysaccharide utilization by the human colonic bacterium, bacteroides intestinalis DSM 17393
Author(s):Vasconcelos Pereira, Gabriel
Director of Research:Cann, Isaac
Doctoral Committee Chair(s):Cann, Isaac
Doctoral Committee Member(s):Mackie, Roderick; Ridlon, Jason; Vanderpool, Cari
Department / Program:Animal Sciences
Discipline:Animal Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Biochemistry, Enzymology, Human Microbiome, Human Gut, Phenolic compounds, Ferulic acid, Esterase, Bacteroides, Fiber degradation, Immunology
Abstract:The human gastrointestinal microbiome has co-evolved with the host for an extended period, generating an intrinsic metabolic network capable of modulating both the microbial community and host physiology. Changes in the gut environment shape the microbial community with one of the main driving characteristics being competition for nutrients. A key aspect for nutrient acquisition in the gut is the ability of the microbes to degrade and ferment dietary fiber. These chemically diverse polysaccharides are the main components of the plant cell wall and function as the primary energy source for the microbiota. The dietary polysaccharides are largely indigestible by the digestive enzymes of the host. The phylum Bacteroidetes, one of the main colonizers of the gastrointestinal tract (GIT), has evolved one of the largest arrays of carbohydrate-associated enzymes (CAZymes) responsible for degrading polysaccharides. Furthermore, these bacteria have highly organized gene clusters, known as polysaccharide utilization loci (PUL), encoding the enzymes required for the degradation and transport of the sugar components of dietary fiber. These PULs also possess an unusual regulatory mechanism found only in Bacteroidetes, similar to the canonical two-component system encoded by organisms in all three domains of life. The two-component system is involved in signal transduction, mediated by a histidine-kinase sensor and a response regulator. Interestingly, the Bacteroidetes encode this system in a single polypeptide that can be delineated into a sensor, a Y_Y_Y, a histidine-kinase, a histidine-ATPase, and response regulator modules, respectively. Through our work, we aim to understand the degradation of esterified arabinoxylan by diverse Bacteroides spp. and to develop a rapid approach for identifying the potential target of a vast number of uncharacterized PULs. Our findings demonstrate that some Bacteroides spp. highly express an esterase-enriched cluster during growth on esterified arabinoxylan compared to the mixture of its component monosaccharides xylose and arabinose. Biochemical analysis of the proteins encoded by the esterase-enriched cluster demonstrated diverse enzymatic activities and substrate specificities capable of working synergistically to fully depolymerize complex arabinoxylan substrates. Interestingly, the hypothetical domain encoded by BACINT_01040 demonstrated one of the most versatile feruloyl esterase activities, and the enzyme was able to cleave the ferulic acid side chain of every substrate tested in our studies. Moreover, we demonstrated that the bacteria harboring the esterase-enriched cluster do not metabolize the ferulic acid during growth on wheat bran. Thus, the plant phenolic compound accumulated in the spent medium. Furthermore, the accumulated ferulic acid was able to modulate the immune system and induce a Th1-type immune response and viral defenses in both gastrointestinal cell lines and mouse model. Due to the vast number of uncharacterized PULs encoded in the microbiome, it is important to develop a rapid means to assign function to these gene clusters. Our current hypothesis relies on the observation of a potential endo-acting enzyme near the susC/susD-like genes. We hypothesized that these enzymes “scout” the environment for their associated target polysaccharides. In this work, we biochemically characterized the activity of two “scouting enzymes” on arabinoxylan and arabinan, thus showing that a PUL-associated hybrid two-component system (HTCS) sensor domain is able to bind and sense the products of its associated scouting enzyme. This hypothesis was further corroborated by transcriptomic analysis of B. intestinalis grown on each polysaccharide compared to its monomeric components. Thus, the results demonstrated in this study may advance the understanding of polysaccharide utilization by Bacteroidetes species by rapidly assigning function to uncharacterized PULs. Further studies may also allow us to identify a polysaccharide-degrading signature by the Bacteroidetes species, which may be used for personalized dietary interventions and modulation of the gut community.
Issue Date:2018-07-23
Type:Thesis
URI:http://hdl.handle.net/2142/102877
Rights Information:Copyright 2018 Gabriel Vasconcelos Pereira
Date Available in IDEALS:2019-02-08
Date Deposited:2018-12


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