Complex glycan utilization preferences of human gut bacteria

Yunus Emre Tuncil, Purdue University

Abstract

Complex glycans, making up dietary fiber, have gained significance in recent years as they are the main energy source for the colonic microbiota which are physiologically important for the host health. Understanding glycan utilization strategies of the members of this community is essential to maximize beneficial health outcomes obtained from them. In order to investigate how the members of our gut microbiota utilize glycans and activate their respective polysaccharide utilization loci (PULs), we performed a series of time-course assays in which two model organisms, Bacteroides thetaiotamicron (Bt) and B. ovatus (Bo), were individually grown in a medium containing carbohydrates utilized by both bacteria: amylopectin (AP), arabinan (ARAB), chondroitin sulphate (CS), pectic galactan (PG), polygalacturonic acid (PGA), and rhamnogalacturonan I (RGI), and measured remaining substrates over time as well as PULs expression. Bt and Bo utilized some glycans before others, but with different prioritizations supporting past work that bacterial species show species-specific hierarchical preference to dietary fibers. Bacteria sensitivities to some glycans are tightly tuned to the residual concentration of polysaccharide in the medium, while others remain highly expressed even after most of the target substrates have been depleted. For example, expression of the PGA utilization gene by Bo gradually reduced as PGA amount decreased in the media; however, expression of the CS utilization gene by Bo remained at a high level even after the substrate was reduced by about 98%. Next, the possibility of change in substrate hierarchy was explored when bacteria are in a competitive environment. Co-culturing of these organisms in the same mixture resulted in reduction in expression of some Bt PULs, however the hierarchical orders remained generally the same. Relative abundances of these species in the co-cultured environment remained constant throughout the exponential phase. This is, most probably, due to their different glycan priorities which allow them to maintain their coexistence. Next, a hypothesis was tested whether molecular structure of a glycan affects its place in the hierarchy. To test this, we repeated the hierarchical substrate preference test for Bo by substituting AP with a starch analog [maltohexaose (MH) (low molecular weight hexasaccharide derived from starch)]. AP was used after RGI by Bo when AP was included in the mixture, whereas MH was used before RGI, so the utilization of RGI by Bo was delayed in the presence of MH. These results provide information about the strategies of Bt and Bo use to utilize different glycans that are often present as a mixture in the colon, and the effects of a particular carbohydrate chemical structure on preference of use. Finally, we questioned whether such a hierarchical preference of glycans also exists in the highly competitive environment of the colon. To answer this, we designed a series of in vitro fermentation studies using fecal microbiota obtained from three healthy individuals, and compared the degradation profiles of single glycans with a mixture containing an equal amount of these substrates. These results showed that, from the polysaccharide perspective, hierarchical preferences exist in the competitive environment of the colon, and degradation of some glycans by fecal microbiota is delayed when they are present as a mixture. Therefore, this might be a logical strategy for delivering particular dietary fibers into the distal part of the colon so that the fermentative activity can be stimulated throughout the colon, and beneficial bacteria as well as more SCFA production in the distal colon can be promoted.

Degree

Ph.D.

Advisors

Hamaker, Purdue University.

Subject Area

Food Science|Microbiology

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