Open Collections

Functional dissection of beta-glucan utilization by prominent human gut symbionts

University of British Columbia

The human gut microbiota (HGM) is a remarkably dense and dynamic community of microbes with incredible collective metabolic capacity. Complex glycans (“dietary fiber”) evade digestion by the human host and feed the HGM, driving its composition, and in turn influencing diverse facets of host health. In order to access these otherwise recalcitrant glycans, Bacteroidetes, a dominant bacterial phylum in the HGM, co-localize genes encoding a membrane-associated machinery that work in concert to bind, break down, and sequester target glycan into co-regulated Polysaccharide Utilization Loci (PULs). One important class of complex glycans with numerous documented health benefits is beta-glucans. In this thesis, I undertake holistic functional characterization, combining biochemistry, structural biology, microbiology, and (meta)genomics, of PULs targeting two distinct subclasses of beta-glucan: mixed-linkage beta-glucan (MLG), and beta(1,3)-glucan. The MLG utilization locus (MLGUL) from B. ovatus, necessary to enable growth on MLG, encodes an endo-beta-glucanase anchored to the cell surface to break down MLG, and a periplasmic exo-beta-glucosidase to completely saccharify the fragments imported by the TonB-dependent outer-membrane transporter. The process is aided by two cell surface glycan-binding proteins (SGBPs) which employ binding platforms shaped to complement that of target MLG to recruit and retain the glycan at cell surface. Growth analysis combined with comparative genomics reveal MLGULs serve as genetic markers for ability to grow on MLG. Metagenomic analysis further suggest MLGUL presence in, and consequent ability to utilize MLG by, the HGM of a majority of humans worldwide. A distinct set of syntenic beta(1,3)-glucan utilization loci (1,3GULs) from three prominent Bacteroides species (B. uniformis, B. thetaiotaomicron, and B. fluxus) were subject to similar holistic functional characterization. Differential ability to grow on beta-glucan congeners is driven by synergy between enzymes and SGBPs: a particular 1,3GUL can mediate utilization of a beta-glucan congener if it encodes both an enzyme that can hydrolyze the target, as well as an SGBP that can bind the target. Detailed structure-function analysis of glycoside hydrolases (GH), including a family-first structure of a GH158, and a suite of SGBPs reveal the molecular basis of catalytic and binding specificities that together give rise to species-differential specificity of 1,3GULs.

Text

2021-04-22

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/77919

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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