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Prevotella copri is a prevalent inhabitant of the human gut and has been associated with plant‐rich diet consumption and diverse health states. The underlying genetic basis of these associations remains enigmatic due to the lack of genetic tools. Here, we developed a novel versatile genetic toolbox for rapid and efficient genetic insertion and allelic exchange applicable to P. copri strains from multiple clades. Enabled by the genetic platform, we systematically investigated the specificity of polysaccharide utilization loci (PULs) and identified four highly conserved PULs for utilizing arabinan, pectic galactan, arabinoxylan, and inulin, respectively. Further genetic and functional analysis of arabinan utilization systems illustrate that P. copri has evolved two distinct types of arabinan‐processing PULs (PULAra) and that the type‐II PULAra is significantly enriched in individuals consuming a vegan diet compared to other diets. In summary, this genetic toolbox will enable functional genetic studies for P. copri in future., A novel conjugation‐based gene insertion/excision system allowing genetic manipulation of the previously intractable bacterium Prevotella copri provides insight into human diet effects on commensal bacterial metabolism.

Prevotella copriis a prevalent inhabitant of the human gut and has been associated with plant-rich diet consumption and diverse health states. The underlying genetic basis of these associations remains enigmatic due to the lack of genetic tools. Here, we developed a novel versatile genetic toolbox for rapid and efficient genetic insertion and allelic exchange applicable toP. copristrains from multiple clades. Enabled by the genetic platform, we systematically investigated the specificity of polysaccharide utilization loci (PULs), and identified four highly conserved PULs for utilizing arabinan, pectic galactan, arabinoxylan and inulin, respectively. Further genetic and functional analysis of arabinan utilization systems illustrate thatP. coprihas evolved two distinct types of arabinan-processing PULs (PULAra) and that the type-II PULArais significantly enriched in individuals consuming a vegan diet compared to other diets. In summary, this genetic toolbox will enable functional genetic studies forP. copriin the future.

CD81 plays a role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus. Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association, but had disparate effects on HCV, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified an allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol unbound) or closed (cholesterol bound) conformation. The open mutant of CD81 exhibited reduced receptor activity whereas the closed mutant was enhanced. These data are consistent with cholesterol switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in health and disease.

Prevotella spp. form one of the most dominant bacterial genera in the human gut. Multiple Prevotella spp. can co-exist in some but not all individuals consuming plant-based diets. To functionally investigate the relationship between Prevotella competition and diet, we isolated several novel Prevotella spp. from the mouse gut, performed genome and metatranscriptome analysis as well as a series of competition experiments under different dietary conditions. These experiments revealed that diverse dominant Prevotella species compete for similar metabolic niches in vivo, which is linked to the upregulation of specific Polysaccharide Utilization Loci (PULs). Complex plant-derived polysaccharides are required, and particularly arabinoxylan is sufficient, for the expansion of Prevotella spp. The most dominant Prevotella species encodes a specific tandem-repeat trsusC/D PUL, which is conserved in highly abundant Prevotella copri strains further expanded in humans consuming a vegan diet. Our data suggest efficient (arabino)xylan-utilization as a factor contributing to Prevotella dominance.

OBJECTIVES: Caloric restriction has been associated with beneficial effects on metabolic diseases such as type 2 diabetes, cardiovascular diseases, and cancer but the mechanisms are diverse and not fully understood. Interactions between the human host and its gut microbiota might play a crucial role in this context, but these have not yet been analysed in detail. Therefore, we conducted a pilot intervention study to investigate how the microbial community in the human gut adapts to caloric restriction. Additionally, we investigated whether the compliance improved if the participants during fasting consumed prebiotics, which are supposed to reduce sensations of hunger while not providing calories through digestion in the small intestine. METHODS: We recruited six healthy volunteers (age: 20–50 years, BMI: 20–25 kg/m(2)) for a pilot intervention study using a randomized crossover design. The study consisted of two sequential fasting periods of which one included additional prebiotics. Participants were fasting for three consecutive days consuming a total of 300 kcal daily provided by vegetable juices, framed by two days with a total daily calorie intake of 800 kcal, respectively. During one of the fasting periods, participants consumed additionally 24 g of inulin daily. Stool samples were collected for the analysis of the microbial composition by 16S rRNA sequencing. RESULTS: We observed no clear change in alpha diversity during the period of caloric reduction. However, quantitative changes in the composition of the microbiome was visible in mainly all participants. Additional intake of the prebiotic inulin did not influence compliance for the fasting intervention. However, we observed a clear reduction in alpha diversity in the gut microbiome after inulin ingestion during the fasting period. CONCLUSIONS: Caloric restriction appears to have no significant influence on the gut microbiome itself but inulin consumption during fasting seems to selectively promote the growth of some bacterial families leading to an overall decrease of alpha diversity in the gut microbiome. Further studies with a larger sample size are warranted to verify our observations. FUNDING SOURCES: Institutional budget, no external funding.