Dietary sphinganine is selectively assimilated by members of the gut microbiome

Abstract

Functions of the gut microbiome have a growing number of implications for host metabolic health, with diet being one of the most significant influences on microbiome composition. Compelling links between diet and the gut microbiome suggest key roles for various macronutrients, including lipids, yet how individual classes of dietary lipids interact with the microbiome remain largely unknown. A class of lipids known as sphingolipids are bioactive components of most foods and are produced by prominent gut microbes. This makes sphingolipids intriguing candidates for shaping diet-microbiome interactions.Here, we use a click-chemistry based approach to track the incorporation of bioorthogonal dietary omegaalkynyl sphinganine (sphinganine alkyne -SAA) into the gut microbial community (Click). Identification of microbe and SAA-specific metabolic products was achieved by fluorescence-based sorting of SAA containing microbes (Sort), 16S rRNA gene sequencing to identify the sphingolipid-interacting microbes (Seq), and comparative metabolomics to identify products of SAA assimilation by the microbiome (Spec). Together this approach, Click-Sort-Seq-Spec (ClickSSS), revealed that SAA-assimilation was nearly exclusively performed by gut Bacteroides, indicating that sphingolipid-producing bacteria play a major role in processing dietary sphinganine. Comparative metabolomics of cecal microbiota from SAAtreated mice showed conversion of SAA to a suite of dihydroceramides, consistent with metabolic activity via Bacteroides and Bifidobacterium. Additionally, other sphingolipid-interacting microbes were identified with a focus on an uncharacterized ability of Bacteroides and Bifidobacterium to metabolize 3 dietary sphingolipids. Therefore, ClickSSS provides a platform to study the flux of virtually any alkynelabeled metabolite in diet-microbiome interactions. 7 specific qualities of the alkyne, SAA-derived metabolites are revealed through the use of liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) (Spec).Application of Click-Sort-Seq-Spec (ClickSSS) (Fig. 1) revealed a set of microbes that take up dietary sphingolipids. This included the sphingolipid-producing Bacteroides and beneficial microbes such as Bifidobacterium that do not produce sphingolipids. Comparative metabolomics revealed the dietdependent sphingolipidome of both Bacteroides thetaiotaomicaron (B. theta) and Bifidobacterium longum subsp. infantis (B. longum). Our analysis suggests that diets rich in sphingolipids can influence the composition of the microbiome with implications for supporting the colonization of beneficial microbes. Moreover, ClickSSS can be applied to a variety of diet-microbiome systems to uncover the mechanism of nutrient interactions with the gut microbial metabolome.8 Materials and Methods General InformationAll buffer, reagents, media, and instrumentation were utilized sterile. All solvents used for metabolomics were purchased from Fisher Scientific as HPLC grade. Bacterial culturingBacteroides thetaiotaomicron strain V...