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Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan-microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health.
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Bifidobacteria are among the earliest colonizers of the human gut, conferring numerous health benefits. While multiple Bifidobacterium strains are used as probiotics, accumulating evidence suggests that the individual responses to probiotic supplementation may vary, likely due to a variety of factors, including strain type(s), gut community composition, dietary habits of the consumer, and other health/lifestyle conditions. Given the saccharolytic nature of bifidobacteria, the carbohydrate composition of the diet is one of the primary factors dictating the colonization efficiency of Bifidobacterium strains. Therefore, a comprehensive understanding of bifidobacterial glycan metabolism at the strain level is necessary to rationally design probiotic or synbiotic formulations that combine bacterial strains with glycans that match their nutrient preferences. In this study, we systematically reconstructed 66 pathways involved in the utilization of mono-, di-, oligo-, and polysaccharides by analyzing the representation of 565 curated metabolic functional roles (catabolic enzymes, transporters, transcriptional regulators) in 2973 non-redundant cultured Bifidobacterium isolates and metagenome-assembled genomes (MAGs). Our analysis uncovered substantial heterogeneity in the predicted glycan utilization capabilities at the species and strain level and revealed the presence of a yet undescribed phenotypically distinct subspecies-level clade within the Bifidobacterium longum species. We also identified Bangladeshi isolates harboring unique gene clusters tentatively implicated in the breakdown of xyloglucan and human milk oligosaccharides. Predicted carbohydrate utilization phenotypes were experimentally characterized and validated. Our large-scale genomic analysis considerably expands the knowledge of carbohydrate metabolism in bifidobacteria and provides a foundation for rationally designing single- or multi-strain probiotic formulations of a given bifidobacterial species as well as synbiotic combinations of bifidobacterial strains matched with their preferred carbohydrate substrates.
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Severe acute malnutrition (SAM), defined anthropometrically as a weight-for-length z-score more than 3 standard deviations below the mean (WLZ<-3), affects 19 million children under 5-years-old worldwide. Complete anthropometric recovery after standard inventions is rare with children often left with moderate acute malnutrition (MAM; WLZ -2 to -3). Here we conduct a randomized controlled trial (RCT), involving 12-18-month-old Bangladeshi children from urban and rural sites, who after hospital-based treatment for SAM received a 3-month intervention with a microbiota-directed complementary food (MDCF-2) or a ready-to-use supplementary food (RUSF) as they transitioned to MAM. The rate of WLZ improvement was significantly greater with MDCF-2 than the more calorically-dense RUSF, as we observed in a previous RCT of Bangladeshi children with MAM without antecedent SAM. A correlated meta-analysis of aptamer-based measurements of 4,520 plasma proteins in this and the prior RCT revealed 215 proteins positively-associated with WLZ (prominently those involved in musculoskeletal and CNS development) and 44 negatively-associated proteins (related to immune activation), with a significant enrichment in levels of the positively WLZ-associated proteins in the MDCF-2 arm. Characterizing changes in 754 bacterial metagenome-assembled genomes in serially collected fecal samples disclosed the effects of acute rehabilitation for SAM on the microbiome, its transition as each child achieves a state of MAM, and how specific strains of Prevotella copri function at the intersection between MDCF-2 glycan metabolism and the rescue of growth faltering. These results provide a rationale for further testing the generalizability of the efficacy of MDCF and identify biomarkers for defining treatment responses.
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The contribution of placental immune responses to congenital Zika virus (ZIKV) syndrome remains poorly understood. Here, we leveraged a mouse model of ZIKV infection to identify mechanisms of innate immune restriction exclusively in the fetal compartment of the placenta. ZIKV principally infected mononuclear trophoblasts in the junctional zone, which was limited by mitochondrial antiviral-signaling protein (MAVS) and type I interferon (IFN) signaling mechanisms. Single nuclear RNA sequencing revealed MAVS-dependent expression of IFN-stimulated genes (ISGs) in spongiotrophoblasts but not in other placental cells that use alternate pathways to induce ISGs. ZIKV infection of Ifnar1-/- or Mavs-/- placentas was associated with greater infection of the adjacent immunocompetent decidua, and heterozygous Mavs+/- or Ifnar1+/- dams carrying immunodeficient fetuses sustained greater maternal viremia and tissue infection than dams carrying wild-type fetuses. Thus, MAVS-IFN signaling in the fetus restricts ZIKV infection in junctional zone trophoblasts, which modulates dissemination and outcome for both the fetus and the pregnant mother.
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Proteínas Adaptadoras Transductoras de Señales , Decidua , Feto , Interferón Tipo I , Placenta , Receptor de Interferón alfa y beta , Transducción de Señal , Trofoblastos , Infección por el Virus Zika , Virus Zika , Femenino , Animales , Embarazo , Interferón Tipo I/metabolismo , Interferón Tipo I/inmunología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Placenta/inmunología , Placenta/virología , Placenta/metabolismo , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virología , Virus Zika/inmunología , Virus Zika/fisiología , Ratones , Decidua/inmunología , Decidua/virología , Decidua/metabolismo , Feto/inmunología , Feto/virología , Trofoblastos/inmunología , Trofoblastos/virología , Trofoblastos/metabolismo , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Inmunidad Innata , Complicaciones Infecciosas del Embarazo/inmunología , Complicaciones Infecciosas del Embarazo/virología , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: A randomized controlled trial in Bangladeshi children aged 12 to 18 months with moderate acute malnutrition found that dietary supplementation with the microbiota-directed complementary food (MDCF-2) significantly improved weight gain and repaired gut microbiota compared to the ready-to-use supplementary food. However, the MDCF-2 formulation was made daily from locally available ingredients and the need for a packaged, nutritionally compliant, and organoleptically acceptable MDCF-2 prototype was essential for future large-scale clinical studies. OBJECTIVE: The study aimed to develop and assess the acceptability of 3 alternative foil-packaged formulations of MDCF-2 in comparison to current MDCF-2. METHODS: Of the 3 packaged formulations, the Jumpstart version was provided in 2 sachets, the other 2 formulations were provided in a retort-stable foil pouch extended by sterilization, and microbiological growth was monitored over 10 months. The acceptability study included 40 children aged 8 to 12 months living in an urban slum in Dhaka, and the organoleptic properties were assessed using a 7-point hedonic scale. RESULTS: In the 100 g distributed over the 2 sessions, children consumed 82.5 ± 7.84 g (mean ± SD) of kitchen-prepared MDCF-2, 85.4 ± 7.15 g of the "Jumpstart" MDCF-2 formulation, 85.4 ± 8.70 g of the MDCF-2 with green banana powder, and 86.2 ± 4.26 g of the MDCF-2 with sweet potato formulation. The "Jumpstart" MDCF-2 and MDCF-2 with sweet potato achieved the highest overall acceptability scores on the hedonic scale; although none of the shelf-stable formulations were significantly different from the kitchen-prepared MDCF-2. CONCLUSIONS: Packaged, shelf-stable prototypes of MDCF-2 exhibited comparable acceptability among Bangladeshi children aged 8 to 12 months to the original freshly prepared formulation.
Plain language titleDevelopment and Acceptability of Shelf-Stable Microbiota-Directed Complementary FoodsPlain language summaryCountries around the world are making progress in fighting malnutrition, but it may be challenging to achieve the global nutrition targets for undernourished children by 2025 at the current rate. To address this problem, we need special types of food that can help children grow, develop properly, and sustain their growth. Children with moderate acute malnutrition have imbalanced gut bacteria. By providing them with the right nutrition, we can restore the healthy bacteria in their gut using a special food called microbiota-directed complementary food (MDCF). Currently, this food is made daily in the icddr, b established field kitchen, which follows standard production measures to control the quality of MDCF preparation, but we need to create stable prototypes that can be stored and used in different settings. This study was designed to develop shelf-stable new formulations in industrial settings and check their acceptability among children with moderate acute malnutrition. The shelf life of the food was extended using a sterilization method, and its microbiological safety was monitored for a year. The actual consumption and acceptability of these foods were evaluated and all these formulations were acceptable by the children and their mother. The children consumed all 4 food formulations in substantial quantities, with consumption rates exceeding 80% for each formulation. This study showed positive results in Bangladesh, but more research is needed to see if these formulations work well in other geographies and over longer periods.
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BACKGROUND: Globally, stunting affects â¼150 million children under five, while wasting affects nearly 50 million. Current interventions have had limited effectiveness in ameliorating long-term sequelae of undernutrition including stunting, cognitive deficits and immune dysfunction. Disrupted development of the gut microbiota has been linked to the pathogenesis of undernutrition, providing potentially new treatment approaches. METHODS: 124 Bangladeshi children with moderate acute malnutrition (MAM) enrolled (at 12-18 months) in a previously reported 3-month RCT of a microbiota-directed complementary food (MDCF-2) were followed for two years. Weight and length were monitored by anthropometry, the abundances of bacterial strains were assessed by quantifying metagenome-assembled genomes (MAGs) in serially collected fecal samples and levels of growth-associated proteins were measured in plasma. FINDINGS: Children who had received MDCF-2 were significantly less stunted during follow-up than those who received a standard ready-to-use supplementary food (RUSF) [linear mixed-effects model, ßtreatment group x study week (95% CI) = 0.002 (0.001, 0.003); P = 0.004]. They also had elevated fecal abundances of Agathobacter faecis, Blautia massiliensis, Lachnospira and Dialister, plus increased levels of a group of 37 plasma proteins (linear model; FDR-adjusted P < 0.1), including IGF-1, neurotrophin receptor NTRK2 and multiple proteins linked to musculoskeletal and CNS development, that persisted for 6-months post-intervention. INTERPRETATION: MDCF-2 treatment of Bangladeshi children with MAM, which produced significant improvements in wasting during intervention, also reduced stunting during follow-up. These results suggest that the effectiveness of supplementary foods for undernutrition may be improved by including ingredients that sponsor healthy microbiota-host co-development. FUNDING: This work was supported by the BMGF (Grants OPP1134649/INV-000247).
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Microbioma Gastrointestinal , Humanos , Lactante , Femenino , Masculino , Bangladesh/epidemiología , Heces/microbiología , Metagenoma , Trastornos del Crecimiento/etiologíaRESUMEN
Environmental enteric dysfunction (EED) is a subclinical syndrome of altered small intestinal function postulated to be an important contributor to childhood undernutrition. The role of small intestinal bacterial communities in the pathophysiology of EED is poorly defined due to a paucity of studies where there has been a direct collection of small intestinal samples from undernourished children. Sixty-three members of a Pakistani cohort identified as being acutely malnourished between 3 and 6 months of age and whose wasting (weight-for-length Z-score [WLZ]) failed to improve after a 2-month nutritional intervention underwent esophagogastroduodenoscopy (EGD). Paired duodenal luminal aspirates and duodenal mucosal biopsies were obtained from 43 children. Duodenal microbiota composition was characterized by sequencing bacterial 16S rRNA gene amplicons. Levels of bacterial taxa (amplicon sequence variants [ASVs]) were referenced to anthropometric indices, histopathologic severity in biopsies, expression of selected genes in the duodenal mucosa, and fecal levels of an immunoinflammatory biomarker (lipocalin-2). A "core" group of eight bacterial ASVs was present in the duodenal samples of 69% of participants. Streptococcus anginosus was the most prevalent, followed by Streptococcus sp., Gemella haemolysans, Streptococcus australis, Granulicatella elegans, Granulicatella adiacens, and Abiotrophia defectiva. At the time of EGD, none of the core taxa were significantly correlated with WLZ. Statistically significant correlations were documented between the abundances of Granulicatella elegans and Granulicatella adiacens and the expression of duodenal mucosal genes involved in immune responses (dual oxidase maturation factor 2, serum amyloid A, and granzyme H). These results suggest that a potential role for members of the oral microbiota in pathogenesis, notably Streptococcus, Gemella, and Granulicatella species, warrants further investigation.IMPORTANCEUndernutrition among women and children is a pressing global health problem. Environmental enteric dysfunction (EED) is a disease of the small intestine (SI) associated with impaired gut mucosal barrier function and reduced capacity for nutrient absorption. The cause of EED is ill-defined. One emerging hypothesis is that alterations in the SI microbiota contribute to EED. We performed a culture-independent analysis of the SI microbiota of a cohort of Pakistani children with undernutrition who had failed a standard nutritional intervention, underwent upper gastrointestinal tract endoscopy, and had histologic evidence of EED in their duodenal mucosal biopsies. The results revealed a shared group of bacterial taxa in their duodenums whose absolute abundances were correlated with levels of the expression of genes in the duodenal mucosa that are involved in inflammatory responses. A number of these bacterial taxa are more typically found in the oral microbiota, a finding that has potential physiologic and therapeutic implications.
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Bacterias , Duodeno , Microbioma Gastrointestinal , ARN Ribosómico 16S , Humanos , Duodeno/microbiología , Duodeno/patología , Femenino , Masculino , ARN Ribosómico 16S/genética , Pakistán , Lactante , Microbioma Gastrointestinal/genética , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Desnutrición/microbiología , Preescolar , Heces/microbiología , Estudios de CohortesRESUMEN
The gut microbiota and tumor-associated macrophages (TAMs) affect tumor responses to anti-programmed cell death protein 1 (PD-1) immune checkpoint blockade. Reprogramming TAM by either blocking or deleting the macrophage receptor triggering receptor on myeloid cells 2 (TREM2) attenuates tumor growth, and lack of functional TREM2 enhances tumor elimination by anti-PD-1. Here, we found that anti-PD-1 treatment combined with TREM2 deficiency in mice induces proinflammatory programs in intestinal macrophages and a concomitant expansion of Ruminococcus gnavus in the gut microbiota. Gavage of wild-type mice with R. gnavus enhanced anti-PD-1-mediated tumor elimination, recapitulating the effect occurring in the absence of TREM2. A proinflammatory intestinal environment coincided with expansion, increased circulation, and migration of TNF-producing CD4+ T cells to the tumor bed. Thus, TREM2 remotely controls anti-PD-1 immune checkpoint blockade through modulation of the intestinal immune environment and microbiota, with R. gnavus emerging as a potential probiotic agent for increasing responsiveness to anti-PD-1.
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Microbioma Gastrointestinal , Inmunoterapia , Macrófagos , Glicoproteínas de Membrana , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1 , Receptores Inmunológicos , Animales , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Ratones , Microbioma Gastrointestinal/inmunología , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Inmunoterapia/métodos , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Macrófagos/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Ratones Noqueados , Femenino , Intestinos/inmunologíaRESUMEN
Interleukin 22 (IL-22) promotes intestinal barrier integrity, stimulating epithelial cells to enact defense mechanisms against enteric infections, including the production of antimicrobial peptides. IL-22 binding protein (IL-22BP) is a soluble decoy encoded by the Il22ra2 gene that decreases IL-22 bioavailability, attenuating IL-22 signaling. The impact of IL-22BP on gut microbiota composition and functioning is poorly understood. We found that Il22ra2-/- mice are better protected against Clostridioides difficile and Citrobacter rodentium infections. This protection relied on IL-22-induced antimicrobial mechanisms before the infection occurred, rather than during the infection itself. Indeed, the gut microbiota of Il22ra2-/- mice mitigated infection of wild-type (WT) mice when transferred via cohousing or by cecal microbiota transplantation. Indicator species analysis of WT and Il22ra2-/- mice with and without cohousing disclosed that IL22BP deficiency yields a gut bacterial composition distinct from that of WT mice. Manipulation of dietary fiber content, measurements of intestinal short-chain fatty acids and oral treatment with acetate disclosed that resistance to C. difficile infection is related to increased production of acetate by Il22ra2-/--associated microbiota. Together, these findings suggest that IL-22BP represents a potential therapeutic target for those at risk for or with already manifest infection with this and perhaps other enteropathogens.
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Citrobacter rodentium , Clostridioides difficile , Infecciones por Enterobacteriaceae , Microbioma Gastrointestinal , Interleucina-22 , Ratones Noqueados , Animales , Ratones , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/prevención & control , Receptores de Interleucina/metabolismo , Receptores de Interleucina/genética , Interleucinas/metabolismo , Ratones Endogámicos C57BL , Infecciones por Clostridium/inmunología , Infecciones por Clostridium/microbiología , Infecciones por Clostridium/prevención & controlRESUMEN
Microbiota-directed complementary food (MDCF) formulations have been designed to repair the gut communities of malnourished children. A randomized controlled trial demonstrated that one formulation, MDCF-2, improved weight gain in malnourished Bangladeshi children compared to a more calorically dense standard nutritional intervention. Metagenome-assembled genomes from study participants revealed a correlation between ponderal growth and expression of MDCF-2 glycan utilization pathways by Prevotella copri strains. To test this correlation, here we use gnotobiotic mice colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains, with or without P. copri isolates closely matching the metagenome-assembled genomes. Combining gut metagenomics and metatranscriptomics with host single-nucleus RNA sequencing and gut metabolomic analyses, we identify a key role of P. copri in metabolizing MDCF-2 glycans and uncover its interactions with other microbes including Bifidobacterium infantis. P. copri-containing consortia mediated weight gain and modulated energy metabolism within intestinal epithelial cells. Our results reveal structure-function relationships between MDCF-2 and members of the gut microbiota of malnourished children with potential implications for future therapies.
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Microbioma Gastrointestinal , Desnutrición , Microbiota , Prevotella , Animales , Ratones , Microbioma Gastrointestinal/genética , Aumento de PesoRESUMEN
Evidence is accumulating that perturbed postnatal development of the gut microbiome contributes to childhood malnutrition1-4. Here we analyse biospecimens from a randomized, controlled trial of a microbiome-directed complementary food (MDCF-2) that produced superior rates of weight gain compared with a calorically more dense conventional ready-to-use supplementary food in 12-18-month-old Bangladeshi children with moderate acute malnutrition4. We reconstructed 1,000 bacterial genomes (metagenome-assembled genomes (MAGs)) from the faecal microbiomes of trial participants, identified 75 MAGs of which the abundances were positively associated with ponderal growth (change in weight-for-length Z score (WLZ)), characterized changes in MAG gene expression as a function of treatment type and WLZ response, and quantified carbohydrate structures in MDCF-2 and faeces. The results reveal that two Prevotella copri MAGs that are positively associated with WLZ are the principal contributors to MDCF-2-induced expression of metabolic pathways involved in utilizing the component glycans of MDCF-2. The predicted specificities of carbohydrate-active enzymes expressed by their polysaccharide-utilization loci are correlated with (1) the in vitro growth of Bangladeshi P. copri strains, possessing varying degrees of polysaccharide-utilization loci and genomic conservation with these MAGs, in defined medium containing different purified glycans representative of those in MDCF-2, and (2) the levels of faecal carbohydrate structures in the trial participants. These associations suggest that identifying bioactive glycan structures in MDCFs metabolized by growth-associated bacterial taxa will help to guide recommendations about their use in children with acute malnutrition and enable the development of additional formulations.
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Alimentos , Microbioma Gastrointestinal , Desnutrición , Polisacáridos , Humanos , Lactante , Bacterias/genética , Bangladesh , Peso Corporal/genética , Heces/microbiología , Microbioma Gastrointestinal/fisiología , Genoma Bacteriano/genética , Desnutrición/microbiología , Metagenoma/genética , Polisacáridos/metabolismo , Aumento de PesoRESUMEN
Understanding how members of the human gut microbiota prioritize nutrient resources is one component of a larger effort to decipher the mechanisms defining microbial community robustness and resiliency in health and disease. This knowledge is foundational for development of microbiota-directed therapeutics. To model how bacteria prioritize glycans in the gut, germfree mice were colonized with 13 human gut bacterial strains, including seven saccharolytic Bacteroidaceae species. Animals were fed a Western diet supplemented with pea fiber. After community assembly, an inducible CRISPR-based system was used to selectively and temporarily reduce the absolute abundance of Bacteroides thetaiotaomicron or B. cellulosilyticus by 10- to 60-fold. Each knockdown resulted in specific, reproducible increases in the abundances of other Bacteroidaceae and dynamic alterations in their expression of genes involved in glycan utilization. Emergence of these "alternate consumers" was associated with preservation of community saccharolytic activity. Using an inducible system for CRISPR base editing in vitro, we disrupted translation of transporters critical for utilizing dietary polysaccharides in Phocaeicola vulgatus, a B. cellulosilyticus knockdown-responsive taxon. In vitro and in vivo tests of the resulting P. vulgatus mutants allowed us to further characterize mechanisms associated with its increased fitness after knockdown. In principle, the approach described can be applied to study utilization of a range of nutrients and to preclinical efforts designed to develop therapeutic strategies for precision manipulation of microbial communities.
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Bacteroides thetaiotaomicron , Bacteroides , Humanos , Animales , Ratones , Bacteroides/genética , Polisacáridos , Bacteroides thetaiotaomicron/genética , Bioensayo , Dieta OccidentalRESUMEN
Preclinical and clinical studies are providing evidence that the healthy growth of infants and children reflects, in part, healthy development of their gut microbiomes1-5. This process of microbial community assembly and functional maturation is perturbed in children with acute malnutrition. Gnotobiotic animals, colonized with microbial communities from children with severe and moderate acute malnutrition, have been used to develop microbiome-directed complementary food (MDCF) formulations for repairing the microbiomes of these children during the weaning period5. Bangladeshi children with moderate acute malnutrition (MAM) participating in a previously reported 3-month-long randomized controlled clinical study of one such formulation, MDCF-2, exhibited significantly improved weight gain compared to a commonly used nutritional intervention despite the lower caloric density of the MDCF6. Characterizing the 'metagenome assembled genomes' (MAGs) of bacterial strains present in the microbiomes of study participants revealed a significant correlation between accelerated ponderal growth and the expression by two Prevotella copri MAGs of metabolic pathways involved in processing of MDCF-2 glycans1. To provide a direct test of these relationships, we have now performed 'reverse translation' experiments using a gnotobiotic mouse model of mother-to-offspring microbiome transmission. Mice were colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains cultured from Bangladeshi infants/children in the study population, with or without P. copri isolates resembling the MAGs. By combining analyses of microbial community assembly, gene expression and processing of glycan constituents of MDCF-2 with single nucleus RNA-Seq and mass spectrometric analyses of the intestine, we establish a principal role for P. copri in mediating metabolism of MDCF-2 glycans, characterize its interactions with other consortium members including Bifidobacterium longum subsp. infantis, and demonstrate the effects of P. copri-containing consortia in mediating weight gain and modulating the activities of metabolic pathways involved in lipid, amino acid, carbohydrate plus other facets of energy metabolism within epithelial cells positioned at different locations in intestinal crypts and villi. Together, the results provide insights into structure/function relationships between MDCF-2 and members of the gut communities of malnourished children; they also have implications for developing future prebiotic, probiotic and/or synbiotic therapeutics for microbiome restoration in children with already manifest malnutrition, or who are at risk for this pervasive health challenge.
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Evidence is accumulating that perturbed postnatal development of the gut microbiome contributes to childhood malnutrition1-4. Designing effective microbiome-directed therapeutic foods to repair these perturbations requires knowledge about how food components interact with the microbiome to alter its expressed functions. Here we use biospecimens from a randomized, controlled trial of a microbiome-directed complementary food prototype (MDCF-2) that produced superior rates of weight gain compared to a conventional ready-to-use supplementary food (RUSF) in 12-18-month-old Bangladeshi children with moderate acute malnutrition (MAM)4. We reconstructed 1000 bacterial genomes (metagenome-assembled genomes, MAGs) present in their fecal microbiomes, identified 75 whose abundances were positively associated with weight gain (change in weight-for-length Z score, WLZ), characterized gene expression changes in these MAGs as a function of treatment type and WLZ response, and used mass spectrometry to quantify carbohydrate structures in MDCF-2 and feces. The results reveal treatment-induced changes in expression of carbohydrate metabolic pathways in WLZ-associated MAGs. Comparing participants consuming MDCF-2 versus RUSF, and MDCF-2-treated children in the upper versus lower quartiles of WLZ responses revealed that two Prevotella copri MAGs positively associated with WLZ were principal contributors to MDCF-2-induced expression of metabolic pathways involved in utilization of its component glycans. Moreover, the predicted specificities of carbohydrate active enzymes expressed by polysaccharide utilization loci (PULs) in these two MAGs correlate with the (i) in vitro growth of Bangladeshi P. copri strains, possessing differing degrees of PUL and overall genomic content similarity to these MAGs, cultured in defined medium containing different purified glycans representative of those in MDCF-2, and (ii) levels of carbohydrate structures identified in feces from clinical trial participants. In the accompanying paper5, we use a gnotobiotic mouse model colonized with age- and WLZ-associated bacterial taxa cultured from this study population, and fed diets resembling those consumed by study participants, to directly test the relationship between P. copri, MDCF-2 glycan metabolism, host ponderal growth responses, and intestinal gene expression and metabolism. The ability to identify bioactive glycan structures in MDCFs that are metabolized by growth-associated bacterial taxa will help guide recommendations about use of this MDCF for children with acute malnutrition representing different geographic locales and ages, as well as enable development of bioequivalent, or more efficacious, formulations composed of culturally acceptable and affordable ingredients.
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Aging is associated with low bone and lean mass as well as alterations in the gut microbiota (GM). In this study, we determined whether the reduced bone mass and relative lean mass observed in old mice could be transferred to healthy young mice by GM transplantation (GMT). GM from old (21-month-old) and young adult (5-month-old) donors was used to colonize germ-free (GF) mice in three separate studies involving still growing 5- or 11-week-old recipients and 17-week-old recipients with minimal bone growth. The GM of the recipient mice was similar to that of the donors, demonstrating successful GMT. GM from old mice did not have statistically significant effects on bone mass or bone strength, but significantly reduced the lean mass percentage of still growing recipient mice when compared with recipients of GM from young adult mice. The levels of propionate in the cecum of mice receiving old donor GM were significantly lower than those in mice receiving young adult donor GM. Bacteroides ovatus was enriched in the microbiota of recipient mice harboring GM from young adult donors. The presence of B. ovatus was not only significantly associated with high lean mass percentage in mice, but also with lean mass adjusted for fat mass in the large human HUNT cohort. In conclusion, GM from old mice reduces lean mass percentage but not bone mass in young, healthy, still growing recipient mice. Future studies are warranted to determine whether GM from young mice improves the musculoskeletal phenotype of frail elderly recipient mice.
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Microbioma Gastrointestinal , Microbiota , Adulto Joven , Humanos , Ratones , Animales , Anciano , Lactante , Trasplante de Microbiota Fecal , Envejecimiento , CiegoRESUMEN
Tau-mediated neurodegeneration is a hallmark of Alzheimer's disease. Primary tauopathies are characterized by pathological tau accumulation and neuronal and synaptic loss. Apolipoprotein E (ApoE)-mediated neuroinflammation is involved in the progression of tau-mediated neurodegeneration, and emerging evidence suggests that the gut microbiota regulates neuroinflammation in an APOE genotype-dependent manner. However, evidence of a causal link between the microbiota and tau-mediated neurodegeneration is lacking. In this study, we characterized a genetically engineered mouse model of tauopathy expressing human ApoE isoforms reared under germ-free conditions or after perturbation of their gut microbiota with antibiotics. Both of these manipulations reduced gliosis, tau pathology, and neurodegeneration in a sex- and ApoE isoform-dependent manner. The findings reveal mechanistic and translationally relevant interrelationships between the microbiota, neuroinflammation, and tau-mediated neurodegeneration.
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Apolipoproteínas E , Microbioma Gastrointestinal , Enfermedades Neuroinflamatorias , Tauopatías , Animales , Humanos , Ratones , Antibacterianos/farmacología , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Modelos Animales de Enfermedad , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/fisiología , Ratones Transgénicos , Enfermedades Neuroinflamatorias/genética , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/microbiología , Proteínas tau/genética , Proteínas tau/metabolismo , Tauopatías/genética , Tauopatías/metabolismo , Tauopatías/microbiología , Factores SexualesRESUMEN
Cellular behaviors emerge from layers of molecular interactions: proteins interact to form complexes, pathways, and phenotypes. We show that hierarchical networks of protein interactions can be defined from the statistical pattern of proteome variation measured across thousands of diverse bacteria and that these networks reflect the emergence of complex bacterial phenotypes. Our results are validated through gene-set enrichment analysis and comparison to existing experimentally derived databases. We demonstrate the biological utility of our approach by creating a model of motility in Pseudomonas aeruginosa and using it to identify a protein that affects pilus-mediated motility. Our method, SCALES (Spectral Correlation Analysis of Layered Evolutionary Signals), may be useful for interrogating genotype-phenotype relationships in bacteria.
Asunto(s)
Mapas de Interacción de Proteínas , Proteoma , Bacterias/genética , Fimbrias Bacterianas , FenotipoRESUMEN
BACKGROUND: Childhood undernutrition is a major public health concern that needs special attention to achieve 2025 global nutrition targets. Moderate acute malnutrition (MAM), manifest as wasting (low weight-for-height), affects 33 million children under 5, yet there are currently no global guidelines for its treatment. We recently performed a randomized-controlled clinical study of a microbiota-directed complementary food formulation (MDCF-2) in 12-18-month-old Bangladeshi children with MAM. The results revealed that MDCF-2, freshly prepared each day, produced a significantly greater improvement in ponderal growth than a standard ready-to-use supplementary food (RUSF), an effect that is associated with repair of the disrupted gut microbial community development that occurs in children with MAM. To test the generalizability of these results in acutely malnourished children at other sites, there is a pressing need for a packaged, shelf-stable, organoleptically-acceptable formulation that is bioequivalent to MDCF-2. This report describes the protocol for a clinical study to evaluate candidate formulations designed to meet these criteria. METHODS: A randomized single-blind study will be conducted in 8-12-month-old Bangladeshi children with MAM to compare the efficacy of alternative shelf-stable MDCF prototypes versus the current MDCF-2 formulation that is produced fresh each day. V4-16S rDNA amplicon and shotgun sequencing datasets will be generated from faecal DNA samples collected from each child enrolled in each group prior to, during, and after treatment to determine the abundances of MDCF-2-responsive bacterial taxa. Efficacy will be assessed by quantifying the change in representation of MDCF-2-responsive gut bacterial taxa after 4-weeks of treatment with freshly prepared MDCF-2 compared to their changes in abundance after treatment with the prototype MDCFs. Equivalence will be defined as the absence of a statistically significant difference, after 4-weeks of treatment, in the representation of faecal bacterial taxa associated with the response to MDCF-2 in participants receiving a test MDCF. DISCUSSION: This trial aims to establish acceptability and equivalence with respect to microbiota repair, of scalable, shelf-stable formulations of MDCF-2 in 8-12-month-old Bangladeshi children with moderate acute malnutrition. TRIAL REGISTRATION: ClinicalTrials.gov (NCT05094024). The trial has been registered before starting enrolment on 23 October 2021.