Spatially resolved lipidomics shows conditional transfer of lipids produced by Bacteroides thetaiotaomicron into the mouse gut.

The extent to which bacterial lipids produced by the gut microbiota penetrate host tissues is unclear. Here, we combined mass spectrometry approaches to identify lipids produced by the human gut symbiont Bacteroides thetaiotaomicron (B. theta) and spatially track these bacterial lipids in the mouse colon. We characterize 130 B. theta lipids by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using wild-type and mutant B. theta strains to confidently identify lipid structures and their interconnected pathways in vitro. Of these, 103 B. theta lipids can be detected and spatially mapped in a single MALDI mass spectrometry imaging run. We map unlabeled bacterial lipids across colon sections of germ-free and specific-pathogen-free (SPF) mice and mice mono-colonized with wild-type or sphingolipid-deficient (BTMUT) B. theta. We observe co-localization of bacterially derived phosphatidic acid with host tissues in BTMUT mice, consistent with lipid penetration into host tissues. These results indicate limited and selective transfer of bacterial lipids to the host.

Characterization of inositol lipid metabolism in gut-associated Bacteroidetes.

Inositol lipids are ubiquitous in eukaryotes and have finely tuned roles in cellular signalling and membrane homoeostasis. In Bacteria, however, inositol lipid production is relatively rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids and sphingolipids, but the pathways remain ambiguous and their prevalence unclear. Here, using genomic and biochemical approaches, we investigated the gene cluster for inositol lipid synthesis in BT using a previously undescribed strain with inducible control of sphingolipid synthesis. We characterized the biosynthetic pathway from myo-inositol-phosphate (MIP) synthesis to phosphoinositol dihydroceramide, determined the crystal structure of the recombinant BT MIP synthase enzyme and identified the phosphatase responsible for the conversion of bacterially-derived phosphatidylinositol phosphate (PIP-DAG) to phosphatidylinositol (PI-DAG). In vitro, loss of inositol lipid production altered BT capsule expression and antimicrobial peptide resistance. In vivo, loss of inositol lipids decreased bacterial fitness in a gnotobiotic mouse model. We identified a second putative, previously undescribed pathway for bacterial PI-DAG synthesis without a PIP-DAG intermediate, common in Prevotella. Our results indicate that inositol sphingolipid production is widespread in host-associated Bacteroidetes and has implications for symbiosis.