UCSF

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. It is sensed by the immune system through Toll-like receptor 4 on innate immune cells, typically indicating to the host that an outside invader is present that needs to be cleared, prompting an inflammatory response. LPS is a potent immunostimulatory molecule, and when this process goes unchecked the consequences can be severe: just nanograms of LPS from a pathogenic bacterium injected into the bloodstream of a mouse will cause the mouse to die of septic shock. Puzzlingly, the human microbiota seems to avoid this response altogether and can be tolerated by the immune system despite producing the same classes of molecules that, when made by pathogens, prompt inflammation. The most prevalent Gram-negative gut bacterial taxon, Bacteroides, makes up ~50% of the cells in a typical Western gut; these cells harbor ~300 mg of LPS, making it one of the highest-abundance molecules in the intestine. However, remarkably little is known about the molecular details of microbiota-immune interactions mediated by Bacteroides LPS. As a starting point for understanding the biological function of Bacteroides LPS, we sought to characterize the biosynthesis and structure of the three classical components of LPS: lipid A, the core oligosaccharide, and the O-antigen repeating unit. We identified genes in Bacteroides thetaiotaomicron involved in the biosynthesis of its lipid A core and glycan, generated mutants that elaborate altered forms of LPS, and used mass spectrometry to interrogate the molecular features of these variants. We demonstrate that the glycan does not appear to have a repeating unit and is therefore better categorized as lipooligosaccharide (LOS) rather than LPS. Our work lays the foundation for developing a structure-function relationship for Bacteroides LPS in the context of host colonization.