Nicholas W. Griffin

Gut microbiota from twins discordant for obesity modulate metabolism in mice.

Introduction Establishing whether specific structural and functional configurations of a human gut microbiota are causally related to a given physiologic or disease phenotype is challenging. Twins discordant for obesity provide an opportunity to examine interrelations between obesity and its associated metabolic disorders, diet, and the gut microbiota. Transplanting the intact uncultured or cultured human fecal microbiota from each member of a discordant twin pair into separate groups of recipient germfree mice permits the donors’ communities to be replicated, differences between their properties to be identified, the impact of these differences on body composition and metabolic phenotypes to be discerned, and the effects of diet-by-microbiota interactions to be analyzed. In addition, cohousing coprophagic mice harboring transplanted microbiota from discordant pairs provides an opportunity to determine which bacterial taxa invade the gut communities of cage mates, how invasion correlates with host phenotypes, and how invasion and microbial niche are affected by human diets. Cohousing Ln and Ob mice prevents increased adiposity in Ob cage mates (Ob). (A) Adiposity change after 10 days of cohousing. *P < 0.05 versus Ob controls (Student’s t test). (B) Bacteroidales from Ln microbiota invade Ob microbiota. Columns show individual mice. Methods Separate groups of germfree mice were colonized with uncultured fecal microbiota from each member of four twin pairs discordant for obesity or with culture collections from an obese (Ob) or lean (Ln) co-twin. Animals were fed a mouse chow low in fat and rich in plant polysaccharides, or one of two diets reflecting the upper or lower tertiles of consumption of saturated fats and fruits and vegetables based on the U.S. National Health and Nutrition Examination Survey (NHANES). Ln or Ob mice were cohoused 5 days after colonization. Body composition changes were defined by quantitative magnetic resonance. Microbiota or microbiome structure, gene expression, and metabolism were assayed by 16S ribosomal RNA profiling, whole-community shotgun sequencing, RNA-sequencing, and mass spectrometry. Host gene expression and metabolism were also characterized. Results and Discussion The intact uncultured and culturable bacterial component of Ob co-twins’ fecal microbiota conveyed significantly greater increases in body mass and adiposity than those of Ln communities. Differences in body composition were correlated with differences in fermentation of short-chain fatty acids (increased in Ln), metabolism of branched-chain amino acids (increased in Ob), and microbial transformation of bile acid species (increased in Ln and correlated with down-regulation of host farnesoid X receptor signaling). Cohousing Ln and Ob mice prevented development of increased adiposity and body mass in Ob cage mates and transformed their microbiota’s metabolic profile to a leanlike state. Transformation correlated with invasion of members of Bacteroidales from Ln into Ob microbiota. Invasion and phenotypic rescue were diet-dependent and occurred with the diet representing the lower tertile of U.S. consumption of saturated fats, and upper tertile of fruits and vegetables, but not with the diet representing the upper tertile of saturated fats, and lower tertile of fruit and vegetable consumption. These results reveal that transmissible and modifiable interactions between diet and microbiota influence host biology. Transforming Fat to Thin How much does the microbiota influence the hosts phenotype? Ridaura et al. (1241214 ; see the Perspective by Walker and Parkhill) obtained uncultured fecal microbiota from twin pairs discordant for body mass and transplanted them into adult germ-free mice. It was discovered that adiposity is transmissible from human to mouse and that it was associated with changes in serum levels of branched-chain amino acids. Moreover, obese-phenotype mice were invaded by members of the Bacteroidales from the lean mice, but, happily, the lean animals resisted invasion by the obese microbiota. Mice carrying gut bacteria from lean humans protect their cage mates from the effects of gut bacteria from fat humans. [Also see Perspective by Walker and Parkhill] The role of specific gut microbes in shaping body composition remains unclear. We transplanted fecal microbiota from adult female twin pairs discordant for obesity into germ-free mice fed low-fat mouse chow, as well as diets representing different levels of saturated fat and fruit and vegetable consumption typical of the U.S. diet. Increased total body and fat mass, as well as obesity-associated metabolic phenotypes, were transmissible with uncultured fecal communities and with their corresponding fecal bacterial culture collections. Cohousing mice harboring an obese twin’s microbiota (Ob) with mice containing the lean co-twin’s microbiota (Ln) prevented the development of increased body mass and obesity-associated metabolic phenotypes in Ob cage mates. Rescue correlated with invasion of specific members of Bacteroidetes from the Ln microbiota into Ob microbiota and was diet-dependent. These findings reveal transmissible, rapid, and modifiable effects of diet-by-microbiota interactions.

Human nutrition, the gut microbiome and the immune system

Marked changes in socio-economic status, cultural traditions, population growth and agriculture are affecting diets worldwide. Understanding how our diet and nutritional status influence the composition and dynamic operations of our gut microbial communities, and the innate and adaptive arms of our immune system, represents an area of scientific need, opportunity and challenge. The insights gleaned should help to address several pressing global health problems.

Interactions between Gut Microbiota, Host Genetics and Diet Modulate the Predisposition to Obesity and Metabolic Syndrome

Obesity, diabetes, and metabolic syndrome result from complex interactions between genetic and environmental factors, including the gut microbiota. To dissect these interactions, we utilized three commonly used inbred strains of mice-obesity/diabetes-prone C57Bl/6J mice, obesity/diabetes-resistant 129S1/SvImJ from Jackson Laboratory, and obesity-prone but diabetes-resistant 129S6/SvEvTac from Taconic-plus three derivative lines generated by breeding these strains in a new, common environment. Analysis of metabolic parameters and gut microbiota in all strains and their environmentally normalized derivatives revealed strong interactions between microbiota, diet, breeding site, and metabolic phenotype. Strain-dependent and strain-independent correlations were found between specific microbiota and phenotypes, some of which could be transferred to germ-free recipient animals by fecal transplantation. Environmental reprogramming of microbiota resulted in 129S6/SvEvTac becoming obesity resistant. Thus, development of obesity/metabolic syndrome is the result of interactions between gut microbiota, host genetics, and diet. In permissive genetic backgrounds, environmental reprograming of microbiota can ameliorate development of metabolic syndrome.

Members of the human gut microbiota involved in recovery from Vibrio cholerae infection

Given the global burden of diarrhoeal diseases, it is important to understand how members of the gut microbiota affect the risk for, course of, and recovery from disease in children and adults. The acute, voluminous diarrhoea caused by Vibrio cholerae represents a dramatic example of enteropathogen invasion and gut microbial community disruption. Here we conduct a detailed time-series metagenomic study of faecal microbiota collected during the acute diarrhoeal and recovery phases of cholera in a cohort of Bangladeshi adults living in an area with a high burden of disease. We find that recovery is characterized by a pattern of accumulation of bacterial taxa that shows similarities to the pattern of assembly/maturation of the gut microbiota in healthy Bangladeshi children. To define the underlying mechanisms, we introduce into gnotobiotic mice an artificial community composed of human gut bacterial species that directly correlate with recovery from cholera in adults and are indicative of normal microbiota maturation in healthy Bangladeshi children. One of the species, Ruminococcus obeum, exhibits consistent increases in its relative abundance upon V. cholerae infection of the mice. Follow-up analyses, including mono- and co-colonization studies, establish that R. obeum restricts V. cholerae colonization, that R. obeum luxS (autoinducer-2 (AI-2) synthase) expression and AI-2 production increase significantly with V. cholerae invasion, and that R. obeum AI-2 causes quorum-sensing-mediated repression of several V. cholerae colonization factors. Co-colonization with V. cholerae mutants discloses that R. obeum AI-2 reduces Vibrio colonization/pathogenicity through a novel pathway that does not depend on the V. cholerae AI-2 sensor, LuxP. The approach described can be used to mine the gut microbiota of Bangladeshi or other populations for members that use autoinducers and/or other mechanisms to limit colonization with V. cholerae, or conceivably other enteropathogens.

Genetic determinants of in vivo fitness and diet responsiveness in multiple human gut Bacteroides

Diet shapes host and gut microbe fitness The human gut microbiota is hugely diverse, with many strain variants having a multiplicity of effects on host metabolism and immunity. To define some of these functions, Wu et al. made libraries of mutants of Bacteroides species known for their capacity to process otherwise intractable dietary fiber. Germ-free mice colonized with defined gut microbiota communities containing the mutants were fed specific diets containing different ratios of fat and fiber. Genes, strains, and species were identified that were associated with specific metabolic pathways. The community responses to dietary shifts were manipulated in an attempt to characterize species for their probiotic or therapeutic potential. Science, this issue 10.1126/science.aac5992> To design probiotics, gut microbe fitness determinants and niches were characterized and responses to dietary changes monitored. INTRODUCTION Relatively little is known about the genetic factors that allow members of the human gut microbiota to occupy their niches. Identification of these factors is important for understanding mechanisms that determine microbiota assembly and perturbation through diet, disease, and clinical treatments. Discovery of these factors should enable new approaches for intervening therapeutically in the functional properties of the human gut microbiota. We present a generalizable approach by which to identify fitness determinants for multiple bacterial strains simultaneously in a model human gut microbiota, obtain gene-level characterization of responses to diet change, and design prebiotics for precision microbiota manipulation. RATIONALE We developed a method—multi-taxon INsertion Sequencing (INSeq)—for monitoring the behavior of tens of thousands of transposon (Tn) mutants of multiple bacterial species and strains simultaneously in the guts of gnotobiotic mice. We focused on four prominent human gut Bacteroides: one strain of B. cellulosilyticus, one strain of B. ovatus, and two strains of B. thetaiotaomicron. INSeq libraries, each composed of 87,000 to 167,000 isogenic Tn mutant strains, were produced (single site of Tn insertion per mutant strain; a total of 11 to 26 Tn insertions represented in the library per gene; and 82 to 92% genes covered per genome). The four mutant libraries were introduced into germ-free mice together with 11 wild-type species commonly present in the human gut microbiota. Animals were given a diet rich in fat and simple sugars but devoid of complex polysaccharides [diet 1 (D1)] or one rich in plant polysaccharides and low in fat (D2), either monotonously or in the sequence D1-D2-D1 or D2-D1-D2. Wecalculated a “fitness index” for each gene on the basis of the relative abundance of its INSeq reads in the fecal or cecal microbiota compared with the input library. In vivo INSeq data were correlated with INSeq data generated from organisms cultured under defined in vitro conditions; microbial RNA-seq profiling of the community’s metatranscriptome; and reconstructions of metabolic pathways, regulons, and polysaccharide utilization loci. On the basis of the results, we designed a prebiotic intervention. RESULTS Multi-taxon INSeq (i) provided a digital readout of the remarkably consistent pattern of community assembly; (ii) identified shared as well as species-, strain-, and diet-specific fitness determinants associated with a variety of metabolic or nutrient processing pathways, including those involving amino acids, carbohydrates, and vitamins/cofactors; (iii) enabled quantitative gene-level measurement of the resilience of the responses to diet perturbations; (iv) revealed that arabinoxylan, the most common hemicellulose in cereals, could be used to deliberately manipulate the representation of Bacteroides cellulosilyticus; and (v) defined the niche adjustments of this and the other Bacteroides to arabinoxylan supplementation of the high-fat diet. CONCLUSION In principle, the approach described can be used to obtain a more comprehensive understanding of how host genotype, diet, physiologic, metabolic, and immune factors, as well as pathologic states, affect niches in gut and nongut habitats, as well as to facilitate development of therapeutic interventions for modifying community structure/function. Identification of a prebiotic that increases the abundance of B. cellulosilyticus. (Left) The four mutant libraries were pooled together with 11 other phylogenetically diverse wild-type strains, and this consortium, representing an artificial human gut microbiota, was introduced into germ-free mice. Community assembly, the effects of diet, and recovery from diet oscillations were characterized at a community, strain, and gene level in these gnotobiotic animals by use of multi-taxon INSeq. (Middle) Multi-taxon INSeq revealed an arabinoxylan utilization locus in B. cellulosilyticus that is critical for the organism’s fitness in the high-fat/simple-sugar diet (D1) context but not in the D2 context. A homologous arabinoxylan utilization locus in B. ovatus was not a fitness determinant with either diet. (Right) Consistent with this finding, supplementation of drinking water with arabinoxylan in mice consuming D1 selectively increased the abundance of B. cellulosilyticus but not B. ovatus. Libraries of tens of thousands of transposon mutants generated from each of four human gut Bacteroides strains, two representing the same species, were introduced simultaneously into gnotobiotic mice together with 11 other wild-type strains to generate a 15-member artificial human gut microbiota. Mice received one of two distinct diets monotonously, or both in different ordered sequences. Quantifying the abundance of mutants in different diet contexts allowed gene-level characterization of fitness determinants, niche, stability, and resilience and yielded a prebiotic (arabinoxylan) that allowed targeted manipulation of the community. The approach described is generalizable and should be useful for defining mechanisms critical for sustaining and/or approaches for deliberately reconfiguring the highly adaptive and durable relationship between the human gut microbiota and host in ways that promote wellness.

The effects of micronutrient deficiencies on bacterial species from the human gut microbiota

Mechanistic studies reveal pronounced effects of vitamin A deficiency on bacterial members of a defined human gut microbiota. A gut bacterial view of micronutrient deficiency Deficiencies in vitamins and minerals (micronutrients) are a global health challenge. In a new study, Hibberd et al. compare the effects of acute dietary deficiency of vitamin A, folate, iron, or zinc in gnotobiotic mice harboring bacterial strains common in the human gut. Vitamin A had the greatest effect on the structure of the bacterial community and gene expression. Bacteroides vulgatus, a bacterial species positively correlated with host growth in gnotobiotic mouse models of postnatal human microbiota development, had the biggest response to vitamin A deficiency, exhibiting an increase in its abundance. Genetic, multi-omic, and pharmacologic analyses indicated that retinol treatment affected B. vulgatus fitness through the activity of the bacterial AcrAB-TolC efflux system. These results suggest that micronutrient imbalances should be considered from the perspective of both the human host and the gut microbiota they possess. Vitamin and mineral (micronutrient) deficiencies afflict 2 billion people. Although the impact of these imbalances on host biology has been studied extensively, much less is known about their effects on the gut microbiota of developing or adult humans. Therefore, we established a community of cultured, sequenced human gut–derived bacterial species in gnotobiotic mice and fed the animals a defined micronutrient-sufficient diet, followed by a derivative diet devoid of vitamin A, folate, iron, or zinc, followed by return to the sufficient diet. Acute vitamin A deficiency had the largest effect on bacterial community structure and metatranscriptome, with Bacteroides vulgatus, a prominent responder, increasing its abundance in the absence of vitamin A. Applying retinol selection to a library of 30,300 B. vulgatus transposon mutants revealed that disruption of acrR abrogated retinol sensitivity. Genetic complementation studies, microbial RNA sequencing, and transcription factor–binding assays disclosed that AcrR is a repressor of an adjacent AcrAB-TolC efflux system. Retinol efflux measurements in wild-type and acrR-mutant strains plus treatment with a pharmacologic inhibitor of the efflux system revealed that AcrAB-TolC is a determinant of retinol and bile acid sensitivity in B. vulgatus. Acute vitamin A deficiency was associated with altered bile acid metabolism in vivo, raising the possibility that retinol, bile acid metabolites, and AcrAB-TolC interact to influence the fitness of B. vulgatus and perhaps other microbiota members. This type of preclinical model can help to develop mechanistic insights about the effects of, and more effective treatment strategies for micronutrient deficiencies.

Effects of a gut pathobiont in a gnotobiotic mouse model of childhood undernutrition

Pathobiont-associated cachexia in a gnotobiotic model of childhood undernutrition is determined by strain-level interactions within the gut microbiota. Neighbors matter A big unanswered question is what determines the effects of enteropathogen burden in children who are undernourished or at risk for undernutrition. In a new study, Wagner and colleagues introduce collections of sequenced gut bacterial strains cultured from healthy or underweight Bangladeshi children into germfree mice fed diets resembling those consumed by the children. The gut bacterial strains were transplanted with or without nontoxigenic or enterotoxigenic Bacteroides fragilis strains. Addition of enterotoxigenic B. fragilis induced cachexia in the transplanted mice, and altered gene expression and metabolic activity of the transplanted bacterial strains. These effects were mitigated by cocolonization with nontoxigenic B. fragilis, illustrating the influence of intra- and interspecies interactions in determining the impact of an enteropathogen on its host. To model how interactions among enteropathogens and gut microbial community members contribute to undernutrition, we colonized gnotobiotic mice fed representative Bangladeshi diets with sequenced bacterial strains cultured from the fecal microbiota of two 24-month-old Bangladeshi children: one healthy and the other underweight. The undernourished donor’s bacterial collection contained an enterotoxigenic Bacteroides fragilis strain (ETBF), whereas the healthy donor’s bacterial collection contained two nontoxigenic strains of B. fragilis (NTBF). Analyses of mice harboring either the unmanipulated culture collections or systematically manipulated versions revealed that ETBF was causally related to weight loss in the context of its native community but not when introduced into the healthy donor’s community. This phenotype was transmissible from the dams to their offspring and was associated with derangements in host energy metabolism manifested by impaired tricarboxylic acid cycle activity and decreased acyl–coenzyme A utilization. NTBF reduced ETBF’s expression of its enterotoxin and mitigated the effects of ETBF on the transcriptomes of other healthy donor community members. These results illustrate how intraspecific (ETBF-NTBF) and interspecific interactions influence the effects of harboring B. fragilis.

Impact of the gut microbiota on enhancer accessibility in gut intraepithelial lymphocytes

Significance Comparing germ-free mice with those colonized at birth or later provides a way to determine how gut microbial community exposure affects the chromatin landscape of cells along the gut or at remote sites, ascertain how alterations in chromatin accessibility are correlated with functional features of different lineages, and determine whether there is a critical window of exposure when microbial signals must be received to alter the landscape durably. Genome-wide analysis of chromatin accessibility in intraepithelial lymphocytes and circulating T cells purified from gnotobiotic mice revealed enhancers and flanking genes involved in signaling and metabolic pathways that are sensitive to colonization status. Colonization does not fundamentally alter lineage-specific cis-regulatory landscapes but induces quantitative changes in the accessibility of preestablished enhancer elements. The gut microbiota impacts many aspects of host biology including immune function. One hypothesis is that microbial communities induce epigenetic changes with accompanying alterations in chromatin accessibility, providing a mechanism that allows a community to have sustained host effects even in the face of its structural or functional variation. We used Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) to define chromatin accessibility in predicted enhancer regions of intestinal αβ+ and γδ+ intraepithelial lymphocytes purified from germ-free mice, their conventionally raised (CONV-R) counterparts, and mice reared germ free and then colonized with CONV-R gut microbiota at the end of the suckling–weaning transition. Characterizing genes adjacent to traditional enhancers and super-enhancers revealed signaling networks, metabolic pathways, and enhancer-associated transcription factors affected by the microbiota. Our results support the notion that epigenetic modifications help define microbial community-affiliated functional features of host immune cell lineages.

Attenuated Effects of Bile Acids on Glucose Metabolism and Insulin Sensitivity in a Male Mouse Model of Prenatal Undernutrition

Prenatal undernutrition and low birth weight are associated with risk of type 2 diabetes and obesity. Prenatal caloric restriction results in low birth weight, glucose intolerance, obesity, and reduced plasma bile acids (BAs) in offspring mice. Because BAs can regulate systemic metabolism and glucose homeostasis, we hypothesized that BA supplementation could prevent diet-induced obesity and glucose intolerance in this model of developmental programming. Pregnant dams were food restricted by 50% from gestational days 12.5 to 18.5. Offspring of both undernourished (UN) and control (C) dams given unrestricted diets were weaned to high-fat diets with or without supplementation with 0.25% w/w ursodeoxycholic acid (UDCA), yielding four experimental groups: C, UN, C + UDCA, and UN + UDCA. Glucose homeostasis, BA composition, liver and intestinal gene expression, and microbiota composition were analyzed in the four groups. Although UDCA supplementation ameliorated diet-induced obesity in C mice, there was no effect in UN mice. UDCA similarly lowered fasting insulin, and improved glucose tolerance, pyruvate tolerance, and liver steatosis in C, but not UN, animals. BA composition differed significantly, and liver and ileal expression of genes involved in BA metabolism (Cyp7b1, Shp) were differentially induced by UDCA in C vs UN animals. Bacterial taxa in fecal microbiota correlated with treatment groups and metabolic parameters. In conclusion, prenatal undernutrition alters responsiveness to the metabolic benefits of BA supplementation, with resistance to the weight-lowering and insulin-sensitizing effects of UDCA supplementation. Our findings suggest that BA metabolism may be a previously unrecognized contributor to developmentally programmed diabetes risk.