No 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 remains largely unknown. Sphingolipids are bioactive components of most foods and are also produced by prominent gut microbes. This makes sphingolipids intriguing candidates for shaping diet–microbiome interactions. Here, we used a click chemistry–based approach to track the incorporation of bioorthogonal dietary omega-alkynyl sphinganine (sphinganine alkyne [SAA]) into the murine gut microbial community (Bioorthogonal labeling). We identified microbial and SAA-specific metabolic products through 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, termed Bioorthogonal labeling-Sort-Seq-Spec (BOSSS), revealed that SAA assimilation is 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 SAA-treated mice revealed conversion of SAA to a suite of dihydroceramides, consistent with metabolic activities of Bacteroides and Bifidobacterium. Additionally, other sphingolipid-interacting microbes were identified with a focus on an uncharacterized ability of Bacteroides and Bifidobacterium to metabolize dietary sphingolipids. We conclude that BOSSS provides a platform to study the flux of virtually any alkyne-labeled metabolite in diet–microbiome interactions.

Consumption of dietary lipids, such as cholesterol, modulates the gut microbiome with consequences for host health through the production of microbiome-derived metabolites. Despite the implications for host metabolism, a limited number of specific interactions of the gut microbiome with diet-derived lipids have been characterized. This is partially because obtaining species-level resolution of the responsible taxa can be challenging and additional approaches are needed to identify health-relevant metabolites produced from cholesterol–microbiome interactions. Here we performed bio-orthogonal labelling sort sequence spectrometry, a click chemistry based workflow, to profile cholesterol-specific host–microbe interactions. Mice were exposed to an alkyne-functionalized variant of cholesterol and 16S ribosomal RNA gene amplicon sequencing of faecal samples identified diet-derived cholesterol-interacting microbes from the genera Bacteroides, Bifidobacterium, Enterococcus and Parabacteroides. Shotgun metagenomic analysis provided species-level resolution of diet-derived cholesterol-interacting microbes with enrichment of bile acid-like and sulfotransferase-like activities. Using untargeted metabolomics, we identify that cholesterol is converted to cholesterol sulfate in a Bacteroides-specific manner via the enzyme BT_0416. Mice monocolonized with Bacteroides thetaiotaomicron lacking Bt_0416 showed altered host cholesterol and cholesterol sulfate compared with wild-type mice, identifying a previously uncharacterized microbiome-transformation of cholesterol and a mechanism for microbiome-dependent contributions to host phenotype. Moreover, identification of a cholesterol-responsive sulfotransferase in Bacteroides suggests diet-dependent mechanisms for altering microbiome-specific cholesterol metabolism. Overall, our work identifies numerous cholesterol-interacting microbes with implications for more precise microbiome-conscious regulation of host cholesterol homeostasis.

No abstract

The aquaculture industry faces growing pressure to reduce the use of antibiotics for control of bacterial diseases. In this study we tested the effectiveness of dietary cecropin A, an insect-derived antimicrobial peptide, at preventing mortality and reducing incidence of carrier status in rainbow trout (Oncorhynchus mykiss) challenged by immersion with Yersinia ruckeri. Additionally, we conducted longitudinal analyses of microbiome changes to elucidate effects of both cecropin A and bacterial infection. An in vitro experiment indicated that Y. ruckeri is susceptible to cecropin A. However, dietary cecropin A did not improve the survival of fish challenged with Y. ruckeri, nor did it decrease the persistence of Y. ruckeri in the intestine of fish that survived infection. Moreover, levels of intestinal Y. ruckeri as measured by qPCR suggested that cecropin A may have negatively impacted the ability of fish to resist colonization by this bacterial pathogen. Concomitantly with the survival experiments, the microbiomes of challenged and mock-challenged fish were sampled at days 0, 3, 8, and 30. The microbiomes were in general dominated by Mycoplasma sp. at days 0, 3 and 8, independent of diet, and whether fish had been challenged or mock-challenged. At day 30, the microbiomes of mock-challenged fish fed the +cecropin diet were characterized by lower internal (alpha) diversity (p<.01), greater relative abundance of Mycoplasma sp., and a decrease in gram-negative taxa, when compared to the microbiomes of fish fed the control diet. The opposite was observed in the microbiome of challenged fish. Lastly, correlation analysis of amplicon sequence variants (ASVs) revealed a negative correlation between the presence of Y. ruckeri and seven ASVs, including Mycoplasma sp., suggesting possible beneficial effects of these taxa. In addition, six ASVs were positively correlated to Y. ruckeri, including Flavobacterium succinicans – a known opportunistic fish pathogen. In conclusion, this study revealed that dietary cecropin A was bioactive and exerted significant effects on the microbiome but did not improve fish resistance to infection by Y. ruckeri. Based on our observations and other published results, it appears that high relative abundance of Mycoplasma sp. correlates with higher resistance to intestinal colonization by bacterial pathogens.