The Biosynthesis of Lipooligosaccharide from Bacteroides thetaiotaomicron

Lipopolysaccharide (LPS), a cell-associated glycolipid that makes up the outer leaflet of the outer membrane of Gram-negative bacteria, is a canonical mediator of microbe-host interactions. The most prevalent Gram-negative gut bacterial taxon, Bacteroides, makes up around 50% of the cells in a typical Western gut; these cells harbor similar to 300 mg of LPS, making it one of the highest-abundance molecules in the intestine. As a starting point for understanding the biological function of Bacteroides LPS, we have identified genes in Bacteroides thetaiotaomicron VPI 5482 involved in the biosynthesis of its lipid A core and glycan, generated mutants that elaborate altered forms of LPS, and used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry to interrogate the molecular features of these variants. We demonstrate, inter alia, that the glycan does not appear to have a repeating unit, and so this strain produces lipooligosaccharide (LOS) rather than LPS. This result contrasts with Bacteroides vulgatus ATCC 8482, which by SDS-PAGE analysis appears to produce LPS with a repeating unit. Additionally, our identification of the B. thetaiotaomicron LOS oligosaccharide gene cluster allowed us to identify similar clusters in other Bacteroides species. Our work lays the foundation for developing a structure-function relationship for Bacteroides LPS/LOS in the context of host colonization. IMPORTANCE Much is known about the bacterial species and genes that make up the human microbiome, but remarkably little is known about the molecular mechanisms through which the microbiota influences host biology. A well-known mechanism by which bacteria influence the host centers around lipopolysaccharide (LPS), a component of the Gram-negative bacterial outer membrane. Pathogen-derived LPS is a potent ligand for host receptor Toll-like receptor 4, which plays an important role in sensing bacteria as part of the innate immune response. Puzzlingly, the most common genus of human gut bacteria, Bacteroides, produces LPS but does not elicit a potent proinflammatory response. Previous work showing that Bacteroides LPS differs structurally from pathogen-derived LPS suggested the outlines of an explanation. Here, we take the next step, elucidating the biosynthetic pathway for Bacteroides LPS and generating mutants in the process that will be of great use in understanding how this molecule modulates the host immune response.