Prebiotics: tools to manipulate the gut microbiome and metabolome.

The human gut is an ecosystem comprising trillions of microbes interacting with the host. The composition of the microbiota and their interactions play roles in different biological processes and in the development of human diseases. Close relationships between dietary modifications, microbiota composition and health status have been established. This review focuses on prebiotics, or compounds which selectively encourage the growth of beneficial bacteria, their mechanisms of action and benefits to human hosts. We also review advances in synthesis technology for human milk oligosaccharides, part of one of the most well-characterized prebiotic-probiotic relationships. Current and future research in this area points to greater use of prebiotics as tools to manipulate the microbial and metabolic diversity of the gut for the benefit of human health.

Hadza Prevotella Require Diet-derived Microbiota Accessible Carbohydrates to Persist in Mice.

Industrialization has transformed the gut microbiota, reducing the prevalence of Prevotella relative to Bacteroides. Here, we isolate Bacteroides and Prevotella strains from the microbiota of Hadza hunter-gatherers of Tanzania, a population with high levels of Prevotella. We demonstrate that plant-derived microbiota-accessible carbohydrates (MACs) are required for persistence of Prevotella copri but not Bacteroides thetaiotaomicron in vivo. Differences in carbohydrate metabolism gene content, expression, and in vitro growth reveal that Hadza Prevotella strains specialize in degrading plant carbohydrates, while Hadza Bacteroides isolates use both plant and host-derived carbohydrates, a difference mirrored in Bacteroides from non-Hadza populations. When competing directly, P. copri requires plant-derived MACs to maintain colonization in the presence of B. thetaiotaomicron, as a no MAC diet eliminates P. copri colonization. Prevotella's reliance on plant-derived MACs and Bacteroides' ability to use host mucus carbohydrates could explain the reduced prevalence of Prevotella in populations consuming a low-MAC, industrialized diet.

Hadza Prevotella require diet-derived microbiota-accessible carbohydrates to persist in mice.

Industrialization has transformed the gut microbiota, reducing the prevalence of Prevotella relative to Bacteroides. Here, we isolate Bacteroides and Prevotella strains from the microbiota of Hadza hunter-gatherers in Tanzania, a population with high levels of Prevotella. We demonstrate that plant-derived microbiota-accessible carbohydrates (MACs) are required for persistence of Prevotella copri but not Bacteroides thetaiotaomicron in vivo. Differences in carbohydrate metabolism gene content, expression, and in vitro growth reveal that Hadza Prevotella strains specialize in degrading plant carbohydrates, while Hadza Bacteroides isolates use both plant and host-derived carbohydrates, a difference mirrored in Bacteroides from non-Hadza populations. When competing directly, P. copri requires plant-derived MACs to maintain colonization in the presence of B. thetaiotaomicron, as a no-MAC diet eliminates P. copri colonization. Prevotella's reliance on plant-derived MACs and Bacteroides' ability to use host mucus carbohydrates could explain the reduced prevalence of Prevotella in populations consuming a low-MAC, industrialized diet.

Proceedings from the 3rd International Conference on Microbiome Engineering.

The human microbiome has been inextricably linked to multiple facets of human physiology. From an engineering standpoint, the ability to precisely control the composition and activity of the microbiome holds great promise for furthering our understanding of disease etiology and for new avenues of therapeutic and diagnostic agents. While the field of microbiome research is still in its infancy, growing engineering efforts are emerging to enable new studies in the microbiome and to rapidly translate these findings to microbiome-based interventions. At the 3rd International Conference on Microbiome Engineering, leading experts in the field presented state-of-the-art work in microbiome engineering, discussing probiotics, prebiotics, engineered microbes, microbially derived biomolecules, and bacteriophage.

Oxidative ornithine metabolism supports non-inflammatory C. difficile colonization.

The enteric pathogen Clostridioides difficile (Cd) is responsible for a toxin-mediated infection that causes more than 200,000 recorded hospitalizations and 13,000 deaths in the United States every year1. However, Cd can colonize the gut in the absence of disease symptoms. Prevalence of asymptomatic colonization by toxigenic Cd in healthy populations is high; asymptomatic carriers are at increased risk of infection compared to noncolonized individuals and may be a reservoir for transmission of Cd infection2,3. Elucidating the molecular mechanisms by which Cd persists in the absence of disease is necessary for understanding pathogenesis and developing refined therapeutic strategies. Here, we show with gut microbiome metatranscriptomic analysis that mice recalcitrant to Cd infection and inflammation exhibit increased community-wide expression of arginine and ornithine metabolic pathways. To query Cd metabolism specifically, we leverage RNA sequencing in gnotobiotic mice infected with two wild-type strains (630 and R20291) and isogenic toxin-deficient mutants of these strains to differentiate inflammation-dependent versus -independent transcriptional states. A single operon encoding oxidative ornithine degradation is consistently upregulated across non-toxigenic Cd strains. Combining untargeted and targeted metabolomics with bacterial and host genetics, we demonstrate that both diet- and host-derived sources of ornithine provide a competitive advantage to Cd, suggesting a mechanism for Cd persistence within a non-inflammatory, healthy gut.

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors.

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of detection and quantification of monosaccharide and linkages. Conventional techniques of glycan analysis include chromatographic/mass-spectrometric methods with throughput on the order of hundreds of samples per day without automation. We demonstrate here, a genetically encoded bacterial biosensor for the high-throughput, linkage-specific detection and quantification of the fucosylated HMO structures, 2'-fucosyllactose and 3-fucosyllactose, which we achieved via heterologous expression of fucosidases. As the presence of lactose in milk or in biotechnological processes could lead to false positives, we also demonstrate the reduction of signal from lactose using different strategies. Due to the high throughput of this technique, many reaction conditions or bioreactor parameters could be assayed in parallel in a matter of hours, allowing for the optimization of HMO manufacturing.

Linkage-Specific Detection and Metabolism of Human Milk Oligosaccharides in Escherichia coli.

Human milk oligosaccharides (HMOs) are important prebiotic complex carbohydrates with demonstrated beneficial effects on the microbiota of neonates. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of monosaccharide and linkage analysis. To enable high-throughput screening of HMO structures, we constructed a whole-cell biosensor that uses heterologous expression of glycosidases to generate linkage-specific, quantitative fluorescent readout for a range of HMOs at detection limits down to 20 µM in approximately 6 hr. We also demonstrate the use of this system for orthogonal control of growth rate or protein expression of particular strains in mixed populations. This work enables rapid non-chromatographic linkage analysis and lays the groundwork for the application of directed evolution to biosynthesis of complex carbohydrates as well as the prebiotic manipulation of population dynamics in natural and engineered microbial communities.