Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A

Abstract

Capsular polysaccharide A (CPSA) is a four-sugar repeating unit polymer found on the surface of the gut symbiont Bacteroides fragilis that has therapeutic potential in animal models of autoimmune disorders. This therapeutic potential has been credited to its zwitterionic character derived from a positively charged N-acetyl-4-aminogalactosamine (AADGal) and a negatively charged 4,6-O-pyruvylated galactose (PyrGal). In this report, using a fluorescent polyisoprenoid chemical probe, the complete enzymatic assembly of the CPSA tetrasaccharide repeat unit is achieved. The proposed pyruvyltransferase, WcfO, galactopyranose mutase, WcfM, and glycosyltransferases, WcfP and WcfN, encoded by the CPSA biosynthesis gene cluster were heterologously expressed and functionally characterized. Pyruvate modification, catalyzed by WcfO, was found to occur on galactose of the polyisoprenoid-linked disaccharide (AADGal-Gal), and did not occur on galactose linked to uridine diphosphate (UDP) or a set of nitrophenyl-galactose analogues. This pyruvate modification was also found to be required for the incorporation of the next sugar in the pathway N-acetylgalactosamine (GalNAc) by the glycosyltransferase WcfP. The pyruvate acetal modification of a galactose has not been previously explored in the context of a polysaccharide biosynthesis pathway, and this work demonstrates the importance of this modification to repeat unit assembly. Upon production of the polyisoprenoid-linked AADGal-PyrGal-GalNAc, the proteins WcfM and WcfN were found to work in concert to form the final tetrasaccharide, where WcfM formed UDP-galactofuranose (Galf) and WcfN transfers Galf to the AADGal-PyrGal-GalNAc. This work demonstrates the first enzymatic assembly of the tetrasaccharide repeat unit of CPSA in a sequential single pot reaction.

Metabolic inhibitors of bacterial glycan biosynthesis

“…H. pylori, C. jejuni, and B. fragilis use some of the same monosaccharide building blocks, yet the structures of the glycans produced by these bacteria and the glycosyltransferases that stitch these higher order glycans together diverge. [79][80][81][82] For example, although H. pylori and C. jejuni have analogous dedicated agellin glycosylation systems, in which pseudaminic acid is installed onto serine and threonine residues on agellin proteins, 1,3 their general glycosylation systems differ. 79,82 These differences have manifested in species-selective utilization of azidosugars in metabolic labeling experiments.…”

Understanding the Holobiont: How Microbial Metabolites Affect Human Health and Shape the Immune System

“…Another important microbial immune modulator is the zwitterionic PSA, produced by the common gut bacterium Bacteroides fragilis (Pantosti et al, 1991; Sharma et al, 2017) (Fig. 3).…”

The hologenome concept of evolution after 10 years

“…For example: (i) production of metabolites from dietary components, such as the conversion of dietary fiber to the short-chain fatty acids, acetate, propionate. and butyrate [ 68 ]; (ii) modification of metabolites that are produced by the host, such as primary bile acids that are converted to secondary bile acids, thus assisting in bile acid recycling [ 69 ]; (iii) de novo synthesis of compounds, such as the important microbial immune modulator polysaccharide A, produced by the common gut bacterium Bacteroides fragilis [ 70 ]; (iv) synthesis of vitamins. Certain gut bacteria can produce vitamin K as well as most of the water-soluble B vitamins [ 71 ].…”