Infant microbiome assembly has been intensely studied in infants from industrialized nations, but little is known about this process in nonindustrialized populations. We deeply sequenced infant stool samples from the Hadza hunter-gatherers of Tanzania and analyzed them in a global meta-analysis. Infant microbiomes develop along lifestyle-associated trajectories, with more than 20% of genomes detected in the Hadza infant gut representing novel species. Industrialized infants-even those who are breastfed-have microbiomes characterized by a paucity of Bifidobacterium infantis and gene cassettes involved in human milk utilization. Strains within lifestyle-associated taxonomic groups are shared between mother-infant dyads, consistent with early life inheritance of lifestyle-shaped microbiomes. The population-specific differences in infant microbiome composition and function underscore the importance of studying microbiomes from people outside of wealthy, industrialized nations.
Gut microbiota metabolism of dietary compounds generates a vast array of microbiome-dependent metabolites (MDMs), which are highly variable between individuals. The uremic MDMs (uMDMs) phenylacetylglutamine (PAG), p-cresol sulfate (PCS), and indoxyl sulfate (IS) accumulate during renal failure and are associated with poor outcomes. Targeted dietary interventions may reduce toxic MDM generation; however, it is unclear if inter-individual differences in diet or gut microbiome dominantly contribute to MDM variance. Here, we use a 7-day homogeneous average American diet to standardize dietary precursor availability in 21 healthy individuals. During dietary homogeneity, the coefficient of variation in PAG, PCS, and IS (primary outcome) did not decrease, nor did inter-individual variation in most identified metabolites; other microbiome metrics showed no or modest responses to the intervention. Host identity and age are dominant contributors to variability in MDMs. These results highlight the potential need to pair dietary modification with microbial therapies to control MDM profiles.
Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.
Gut inflammation directly impacts the growth and stability of commensal gut microbes and can lead to long-lasting changes in microbiota composition that can prolong or exacerbate disease states. While mouse models are used extensively to investigate the interplay between microbes and the inflamed state, the paucity of cultured mouse gut microbes has hindered efforts to determine causal relationships. To address this issue, we are assembling the Collection of Inflammation-Associated Mouse Intestinal Bacteria (CIAMIB). The initial release of this collection comprises 41 isolates of 39 unique bacterial species, covering 4 phyla and containing 10 previously uncultivated isolates, including 1 novel family and 7 novel genera. The collection significantly expands the number of available Muribaculaceae, Lachnospiraceae, and Coriobacteriaceae isolates and includes microbes from genera associated with inflammation, such as Prevotella and Klebsiella. We characterized the growth of CIAMIB isolates across a diverse range of nutritional conditions and predicted their metabolic potential and anaerobic fermentation capacity based on the genomes of these isolates. We also provide the first metabolic analysis of species within the genus Adlercreutzia, revealing these representatives to be nitrate-reducing and severely restricted in their ability to grow on carbohydrates. CIAMIB isolates are fully sequenced and available to the scientific community as a powerful tool to study host-microbiota interactions. IMPORTANCE Attempts to explore the role of the microbiota in animal physiology have resulted in large-scale efforts to cultivate the thousands of microbes that are associated with humans. In contrast, relatively few lab mouse-associated bacteria have been isolated, despite the fact that the overwhelming number of studies on the microbiota use laboratory mice that are colonized with microbes that are quite distinct from those in humans. Here, we report the results of a large-scale isolation of bacteria from the intestines of laboratory mice either prone to or suffering from gut inflammation. This collection comprises dozens of novel isolates, many of which represent the only cultured representatives of their genus or species. We report their basic growth characteristics and genomes and are making them widely available to the greater research community.
The gut microbiome has been identified as a key to immune and metabolic health, especially in industrialized populations. Non-industrialized individuals harbor more diverse microbiomes and distinct bacterial lineages, but systemic under-sampling has hindered insight into the extent and functional consequences of these differences. Here, we performed ultra-deep metagenomic sequencing and laboratory strain isolation on fecal samples from the Hadza, hunter-gatherers in Tanzania, and comparative populations in Nepal and California. We recover 94,971 total genomes of bacteria, archaea, bacteriophage, and eukaryotes, and find that 43% are novel upon aggregating with existing unified datasets. Analysis of in situ growth rates, genetic pN/pS signatures, and high-resolution strain tracking reveal dynamics in the hunter-gatherer gut microbiome that are distinct from industrialized populations. Industrialized versus Hadza gut microbes are enriched in genes associated with oxidative stress, possibly a result of microbiome adaptation to inflammatory processes. We use phylogenomics to reveal that global spread of the spirochaete Treponema succinifaciens parallels historic human migration prior to its extinction in industrialized populations. When combined with a detailed definition of gut-resident strains that are vanishing in industrialized populations, our data demonstrate extensive perturbation in many facets of the gut microbiome brought on by the industrialized lifestyle.
Bacteriophages (phages) are diverse and abundant constituents of microbial communities worldwide, capable of modulating bacterial populations in diverse ways. Here, we describe the phage HNL01, which infects the marine bacterium Vibrio fischeri. We use culture-based approaches to demonstrate that mutations in the exopolysaccharide locus of V. fischeri render this bacterium resistant to infection by HNL01, highlighting the extracellular matrix as a key determinant of HNL01 infection. Additionally, using the natural symbiosis between V. fischeri and the squid Euprymna scolopes, we show that, during colonization, V. fischeri is protected from phages present in the ambient seawater. Taken together, these findings shed light on independent yet synergistic host- and bacterium-based strategies for resisting symbiosis-disrupting phage predation, and we present important implications for understanding these strategies in the context of diverse host-associated microbial ecosystems.
Dietary fibers act through the microbiome to improve cardiovascular health and prevent metabolic disorders and cancer. To understand the health benefits of dietary fiber supplementation, we investigated two popular purified fibers, arabinoxylan (AX) and long-chain inulin (LCI), and a mixture of five fibers. We present multiomic signatures of metabolomics, lipidomics, proteomics, metagenomics, a cytokine panel, and clinical measurements on healthy and insulin-resistant participants. Each fiber is associated with fiber-dependent biochemical and microbial responses. AX consumption associates with a significant reduction in LDL and an increase in bile acids, contributing to its observed cholesterol reduction. LCI is associated with an increase in Bifidobacterium. However, at the highest LCI dose, there is increased inflammation and elevation in the liver enzyme alanine aminotransferase. This study yields insights into the effects of fiber supplementation and the mechanisms behind fiber-induced cholesterol reduction, and it shows effects of individual, purified fibers on the microbiome.
Efforts to probe the role of the gut microbiota in disease would benefit from a system in which patient-derived bacterial communities can be studied at scale. We addressed this by validating a strategy to propagate phylogenetically complex, diverse, stable, and highly reproducible stool-derived communities in vitro. We generated hundreds of in vitro communities cultured from diverse stool samples in various media; certain media generally preserved inoculum composition, and inocula from different subjects yielded source-specific community compositions. Upon colonization of germ-free mice, community composition was maintained, and the host proteome resembled the host from which the community was derived. Treatment with ciprofloxacin in vivo increased susceptibility to Salmonella invasion in vitro, and the in vitro response to ciprofloxacin was predictive of compositional changes observed in vivo, including the resilience and sensitivity of each Bacteroides species. These findings demonstrate that stool-derived in vitro communities can serve as a powerful system for microbiota research.
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 year. 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 infection. 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.
The particularly interdisciplinary nature of human microbiome research makes the organization and reporting of results spanning epidemiology, biology, bioinformatics, translational medicine and statistics a challenge. Commonly used reporting guidelines for observational or genetic epidemiology studies lack key features specific to microbiome studies. Therefore, a multidisciplinary group of microbiome epidemiology researchers adapted guidelines for observational and genetic studies to culture-independent human microbiome studies, and also developed new reporting elements for laboratory, bioinformatics and statistical analyses tailored to microbiome studies. The resulting tool, called 'Strengthening The Organization and Reporting of Microbiome Studies' (STORMS), is composed of a 17-item checklist organized into six sections that correspond to the typical sections of a scientific publication, presented as an editable table for inclusion in supplementary materials. The STORMS checklist provides guidance for concise and complete reporting of microbiome studies that will facilitate manuscript preparation, peer review, and reader comprehension of publications and comparative analysis of published results.
Due to limitations on high-resolution strain tracking, selection dynamics during gut microbiota colonization and transmission between hosts remain mostly mysterious. Here, we introduced hundreds of barcoded Escherichia coli strains into germ-free mice and quantified strain-level dynamics and metagenomic changes. Mutations in genes involved in motility and metabolite utilization are reproducibly selected within days. Even with rapid selection, coprophagy enforced similar barcode distributions across co-housed mice. Whole-genome sequencing of hundreds of isolates revealed linked alleles that demonstrate between-host transmission. A population-genetics model predicts substantial fitness advantages for certain mutants and that migration accounted for ∼10% of the resident microbiota each day. Treatment with ciprofloxacin suggests interplay between selection and transmission. While initial colonization was mostly uniform, in two mice a bottleneck reduced diversity and selected for ciprofloxacin resistance in the absence of drug. These findings highlight the interplay between environmental transmission and rapid, deterministic selection during evolution of the intestinal microbiota.
Gut microorganisms modulate host phenotypes and are associated with numerous health effects in humans, ranging from host responses to cancer immunotherapy to metabolic disease and obesity. However, difficulty in accurate and high-throughput functional analysis of human gut microorganisms has hindered efforts to define mechanistic connections between individual microbial strains and host phenotypes. One key way in which the gut microbiome influences host physiology is through the production of small molecules, yet progress in elucidating this chemical interplay has been hindered by limited tools calibrated to detect the products of anaerobic biochemistry in the gut. Here we construct a microbiome-focused, integrated mass-spectrometry pipeline to accelerate the identification of microbiota-dependent metabolites in diverse sample types. We report the metabolic profiles of 178 gut microorganism strains using our library of 833 metabolites. Using this metabolomics resource, we establish deviations in the relationships between phylogeny and metabolism, use machine learning to discover a previously undescribed type of metabolism in Bacteroides, and reveal candidate biochemical pathways using comparative genomics. Microbiota-dependent metabolites can be detected in diverse biological fluids from gnotobiotic and conventionally colonized mice and traced back to the corresponding metabolomic profiles of cultured bacteria. Collectively, our microbiome-focused metabolomics pipeline and interactive metabolomics profile explorer are a powerful tool for characterizing microorganisms and interactions between microorganisms and their host.
Diet modulates the gut microbiome, which in turn can impact the immune system. Here, we determined how two microbiota-targeted dietary interventions, plant-based fiber and fermented foods, influence the human microbiome and immune system in healthy adults. Using a 17-week randomized, prospective study (n = 18/arm) combined with -omics measurements of microbiome and host, including extensive immune profiling, we found diet-specific effects. The high-fiber diet increased microbiome-encoded glycan-degrading carbohydrate active enzymes (CAZymes) despite stable microbial community diversity. Although cytokine response score (primary outcome) was unchanged, three distinct immunological trajectories in high-fiber consumers corresponded to baseline microbiota diversity. Alternatively, the high-fermented-food diet steadily increased microbiota diversity and decreased inflammatory markers. The data highlight how coupling dietary interventions to deep and longitudinal immune and microbiome profiling can provide individualized and population-wide insight. Fermented foods may be valuable in countering the decreased microbiome diversity and increased inflammation pervasive in industrialized society.
Several enteric pathogens can gain specific metabolic advantages over other members of the microbiota by inducing host pathology and inflammation. The pathogen Clostridium difficile is responsible for a toxin-mediated colitis that causes 450,000 infections and 15,000 deaths in the United States each year; however, the molecular mechanisms by which C. difficile benefits from this pathology remain unclear. To understand how the metabolism of C. difficile adapts to the inflammatory conditions that its toxins induce, here we use RNA sequencing to define, in a mouse model, the metabolic states of wild-type C. difficile and of an isogenic mutant that lacks toxins. By combining bacterial and mouse genetics, we demonstrate that C. difficile uses sorbitol derived from both diet and host. Host-derived sorbitol is produced by the enzyme aldose reductase, which is expressed by diverse immune cells and is upregulated during inflammation-including during toxin-mediated disease induced by C. difficile. This work highlights a mechanism by which C. difficile can use a host-derived nutrient that is generated during toxin-induced disease by an enzyme that has not previously been associated with infection.
Recent studies emphasize the role of microbial metabolites in regulating gastrointestinal (GI) physiology through activation of host receptors, highlighting the potential for inter-kingdom signaling in treating GI disorders. In this study, we show that tryptamine, a tryptophan-derived bacterial metabolite, stimulates mucus release from goblet cells via activation of G-protein-coupled receptor (GPCR) 5-HT4R. Germ-free mice colonized with engineered Bacteroides thetaiotaomicron optimized to produce tryptamine (Trp D+) exhibit decreased weight loss and increased mucus release following dextran sodium sulfate treatment when compared with mice colonized with control B. thetaiotaomicron (Trp D-). Additional beneficial effects in preventing barrier disruption and lower disease activity index were seen only in female mice, highlighting sex-specific effects of the bacterial metabolite. This study demonstrates potential for the precise modulation of mucus release by microbially produced 5-HT4 GPCR agonist as a therapeutic strategy to treat inflammatory conditions of the GI tract.
Ha and colleagues describe a previously unappreciated diversity of microbes in the mesenteric adipose tissue (MAT) surrounding the GI tract. Viable bacteria that are mislocalized from the gut microbiota and metabolically adapted to the MAT contribute to the "creeping fat" of Crohn's disease.
Colon mucus segregates the intestinal microbiota from host tissues, but how it organizes to function throughout the colon is unclear. In mice, we found that colon mucus consists of two distinct O-glycosylated entities of Muc2: a major form produced by the proximal colon, which encapsulates the fecal material including the microbiota, and a minor form derived from the distal colon, which adheres to the major form. The microbiota directs its own encapsulation by inducing Muc2 production from proximal colon goblet cells. In turn, O-glycans on proximal colon-derived Muc2 modulate the structure and function of the microbiota as well as transcription in the colon mucosa. Our work shows how proximal colon control of mucin production is an important element in the regulation of host-microbiota symbiosis.
Immune checkpoint-blocking antibodies that attenuate immune tolerance have been used to effectively treat cancer, but they can also trigger severe immune-related adverse events. Previously, we found that Bifidobacterium could mitigate intestinal immunopathology in the context of CTLA-4 blockade in mice. Here we examined the mechanism underlying this process. We found that Bifidobacterium altered the composition of the gut microbiota systematically in a regulatory T cell (Treg)-dependent manner. Moreover, this altered commensal community enhanced both the mitochondrial fitness and the IL-10-mediated suppressive functions of intestinal Tregs, contributing to the amelioration of colitis during immune checkpoint blockade.
The gut microbiome has been implicated in multiple human chronic gastrointestinal (GI) disorders. Determining its mechanistic role in disease has been difficult due to apparent disconnects between animal and human studies and lack of an integrated multi-omics view of disease-specific physiological changes. We integrated longitudinal multi-omics data from the gut microbiome, metabolome, host epigenome, and transcriptome in the context of irritable bowel syndrome (IBS) host physiology. We identified IBS subtype-specific and symptom-related variation in microbial composition and function. A subset of identified changes in microbial metabolites correspond to host physiological mechanisms that are relevant to IBS. By integrating multiple data layers, we identified purine metabolism as a novel host-microbial metabolic pathway in IBS with translational potential. Our study highlights the importance of longitudinal sampling and integrating complementary multi-omics data to identify functional mechanisms that can serve as therapeutic targets in a comprehensive treatment strategy for chronic GI diseases. VIDEO ABSTRACT.
Despite the rising popularity of plant-based alternative meats, there is limited evidence of the health effects of these products. We aimed to compare the effect of consuming plant-based alternative meat (Plant) as opposed to animal meat (Animal) on health factors. The primary outcome was fasting serum trimethylamine-N-oxide (TMAO). Secondary outcomes included fasting insulin-like growth factor 1, lipids, glucose, insulin, blood pressure, and weight. SWAP-MEAT (The Study With Appetizing Plantfood-Meat Eating Alternatives Trial) was a single-site, randomized crossover trial with no washout period. Participants received Plant and Animal products, dietary counseling, lab assessments, microbiome assessments (16S), and anthropometric measurements. Participants were instructed to consume ≥2 servings/d of Plant compared with Animal for 8 wk each, while keeping all other foods and beverages as similar as possible between the 2 phases. 0.001) were lower during the Plant phase. Among generally healthy adults, contrasting Plant with Animal intake, while keeping all other dietary components similar, the Plant products improved several cardiovascular disease risk factors, including TMAO; there were no adverse effects on risk factors from the Plant products.This trial was registered at clinicaltrials.gov as NCT03718988.
Our emerging view of the gut microbiome largely focuses on bacteria, while less is known about other microbial components, such as bacteriophages (phages). Though phages are abundant in the gut, very few phages have been isolated from this ecosystem. Here, we report the genomes of 27 phages from the United States and Bangladesh that infect the prevalent human gut bacterium Bacteroides thetaiotaomicron. These phages are mostly distinct from previously sequenced phages with the exception of two, which are crAss-like phages. We compare these isolates to existing human gut metagenomes, revealing similarities to previously inferred phages and additional unexplored phage diversity. Finally, we use host tropisms of these phages to identify alleles of phage structural genes associated with infectivity. This work provides a detailed view of the gut's "viral dark matter" and a framework for future efforts to further integrate isolation- and sequencing-focused efforts to understand gut-resident phages.
Stool-based proteomics is capable of significantly augmenting our understanding of host-gut microbe interactions. However, compared to competing technologies, such as metagenomics and 16S rRNA sequencing, it is underutilized due to its low throughput and the negative impact sample contaminants can have on highly sensitive mass spectrometry equipment. Here, we present a new stool proteomic processing pipeline that addresses these shortcomings in a highly reproducible and quantitative manner. Using this method, 290 samples from a dietary intervention study were processed in approximately 1.5 weeks, largely done by a single researcher. These data indicated a subtle but distinct monotonic increase in the number of significantly altered proteins between study participants on fiber- or fermented food-enriched diets. Lastly, we were able to classify study participants based on their diet-altered proteomic profiles and demonstrated that classification accuracies of up to 89% could be achieved by increasing the number of subjects considered. Taken together, this study represents the first high-throughput proteomic method for processing stool samples in a technically reproducible manner and has the potential to elevate stool-based proteomics as an essential tool for profiling host-gut microbiome interactions in a clinical setting. IMPORTANCE Widely available technologies based on DNA sequencing have been used to describe the kinds of microbes that might correlate with health and disease. However, mechanistic insights might be best achieved through careful study of the dynamic proteins at the interface between the foods we eat, our microbes, and ourselves. Mass spectrometry-based proteomics has the potential to revolutionize our understanding of this complex system, but its application to clinical studies has been hampered by low-throughput and laborious experimentation pipelines. In response, we developed SHT-Pro, the first high-throughput pipeline designed to rapidly handle large stool sample sets. With it, a single researcher can process over one hundred stool samples per week for mass spectrometry analysis, conservatively approximately 10× to 100× faster than previous methods, depending on whether isobaric labeling is used or not. Since SHT-Pro is fairly simple to implement using commercially available reagents, it should be easily adaptable to large-scale clinical studies.
A variety of cell surface structures dictate interactions between bacteria and their environment, including their viruses (bacteriophages). Members of the human gut Bacteroidetes characteristically produce several phase-variable capsular polysaccharides (CPSs), but their contributions to bacteriophage interactions are unknown. To begin to understand how CPSs have an impact on Bacteroides-phage interactions, we isolated 71 Bacteroides thetaiotaomicron-infecting bacteriophages from two locations in the United States. Using B. thetaiotaomicron strains that express defined subsets of CPSs, we show that CPSs dictate host tropism for these phages and that expression of non-permissive CPS variants is selected under phage predation, enabling survival. In the absence of CPSs, B. thetaiotaomicron escapes bacteriophage predation by altering expression of eight distinct phase-variable lipoproteins. When constitutively expressed, one of these lipoproteins promotes resistance to multiple bacteriophages. Our results reveal important roles for Bacteroides CPSs and other cell surface structures that allow these bacteria to persist under bacteriophage predation, and hold important implications for using bacteriophages therapeutically to target gut symbionts.
Clostridium difficile infection (CDI) is an enteric bacterial disease that is increasing in prevalence worldwide. C. difficile capitalizes on gut inflammation and microbiome dysbiosis to establish infection, with symptoms ranging from watery diarrhea to toxic megacolon. We reported that the safe-in-human clinical drug ebselen (ClinicalTrials.gov: NCT03013400, NCT01452607, NCT00762671, and NCT02603081) has biochemical, cell-based, and in vivo efficacy against the toxins of C. difficile. Here, we show that ebselen treatment reduces recurrence rates and decreases colitis in a hamster model of relapsing CDI. Furthermore, ebselen treatment does not alter microbiome diversity and promotes recovery back to that of healthy controls after antibiotic-induced dysbiosis in healthy and C. difficile-infected mice. This increased microbiome recovery upon ebselen treatment correlates with a decrease in host-derived inflammatory markers, suggesting that the anti-inflammatory properties of ebselen, combined with its anti-toxin function, help to mitigate the major clinical challenges of CDI, including recurrence, microbial dysbiosis, and colitis.
With the rising rates of obesity and associated metabolic disorders, there is a growing need for effective long-term weight-loss strategies, coupled with an understanding of how they interface with human physiology. Interest is growing in the potential role of gut microbes as they pertain to responses to different weight-loss diets; however, the ways that diet, the gut microbiota, and long-term weight loss influence one another is not well understood. Our primary objective was to determine if baseline microbiota composition or diversity was associated with weight-loss success. A secondary objective was to track the longitudinal associations of changes to lower-carbohydrate or lower-fat diets and concomitant weight loss with the composition and diversity of the gut microbiota. We used 16S ribosomal RNA gene amplicon sequencing to profile microbiota composition over a 12-mo period in 49 participants as part of a larger randomized dietary intervention study of participants consuming either a healthy low-carbohydrate or a healthy low-fat diet. While baseline microbiota composition was not predictive of weight loss, each diet resulted in substantial changes in the microbiota 3-mo after the start of the intervention; some of these changes were diet specific (14 taxonomic changes specific to the healthy low-carbohydrate diet, 12 taxonomic changes specific to the healthy low-fat diet) and others tracked with weight loss (7 taxonomic changes in both diets). After these initial shifts, the microbiota returned near its original baseline state for the remainder of the intervention, despite participants maintaining their diet and weight loss for the entire study. These results suggest a resilience to perturbation of the microbiota's starting profile. When considering the established contribution of obesity-associated microbiotas to weight gain in animal models, microbiota resilience may need to be overcome for long-term alterations to human physiology. This trial was registered at clinicaltrials.gov as NCT01826591.
Secondary bile acids (SBAs) are derived from primary bile acids (PBAs) in a process reliant on biosynthetic capabilities possessed by few microbes. To evaluate the role of BAs in intestinal inflammation, we performed metabolomic, microbiome, metagenomic, and transcriptomic profiling of stool from ileal pouches (surgically created resevoirs) in colectomy-treated patients with ulcerative colitis (UC) versus controls (familial adenomatous polyposis [FAP]). We show that relative to FAP, UC pouches have reduced levels of lithocholic acid and deoxycholic acid (normally the most abundant gut SBAs), genes required to convert PBAs to SBAs, and Ruminococcaceae (one of few taxa known to include SBA-producing bacteria). In three murine colitis models, SBA supplementation reduces intestinal inflammation. This anti-inflammatory effect is in part dependent on the TGR5 bile acid receptor. These data suggest that dysbiosis induces SBA deficiency in inflammatory-prone UC patients, which promotes a pro-inflammatory state within the intestine that may be treated by SBA restoration.
Consumption of glucosinolates, pro-drug-like metabolites abundant in Brassica vegetables, has been associated with decreased risk of certain cancers. Gut microbiota have the ability to metabolize glucosinolates, generating chemopreventive isothiocyanates. Here, we identify a genetic and biochemical basis for activation of glucosinolates to isothiocyanates by Bacteroides thetaiotaomicron, a prominent gut commensal species. Using a genome-wide transposon insertion screen, we identified an operon required for glucosinolate metabolism in B. thetaiotaomicron. Expression of BT2159-BT2156 in a non-metabolizing relative, Bacteroides fragilis, resulted in gain of glucosinolate metabolism. We show that isothiocyanate formation requires the action of BT2158 and either BT2156 or BT2157 in vitro. Monocolonization of mice with mutant BtΔ2157 showed reduced isothiocyanate production in the gastrointestinal tract. These data provide insight into the mechanisms by which a common gut bacterium processes an important dietary nutrient.
Intestinal microbiotas contain beneficial microorganisms that protect against pathogen colonization; treatment with antibiotics disrupts the microbiota and compromises colonization resistance. Here, we determine the impact of exchanging microorganisms between hosts on resilience to the colonization of invaders after antibiotic-induced dysbiosis. We assess the functional consequences of dysbiosis using a mouse model of colonization resistance against Escherichia coli. Antibiotics caused stochastic loss of members of the microbiota, but the microbiotas of co-housed mice remained more similar to each other compared with the microbiotas among singly housed animals. Strikingly, co-housed mice maintained colonization resistance after treatment with antibiotics, whereas most singly housed mice were susceptible to E. coli. The ability to retain or share the commensal Klebsiella michiganensis, a member of the Enterobacteriaceae family, was sufficient for colonization resistance after treatment with antibiotics. K. michiganensis generally outcompeted E. coli in vitro, but in vivo administration of galactitol-a nutrient that supports the growth of only E. coli-to bi-colonized gnotobiotic mice abolished the colonization-resistance capacity of K. michiganensis against E. coli, supporting the idea that nutrient competition is the primary interaction mechanism. K. michiganensis also hampered colonization of the pathogen Salmonella, prolonging host survival. Our results address functional consequences of the stochastic effects of microbiota perturbations, whereby microbial transmission through host interactions can facilitate reacquisition of beneficial commensals, minimizing the negative impact of antibiotics.
The gut microbiota produce hundreds of molecules that are present at high concentrations in the host circulation. Unraveling the contribution of each molecule to host biology remains difficult. We developed a system for constructing clean deletions in Clostridium spp., the source of many molecules from the gut microbiome. By applying this method to the model commensal organism Clostridium sporogenes, we knocked out genes for 10 C. sporogenes-derived molecules that accumulate in host tissues. In mice colonized by a C. sporogenes for which the production of branched short-chain fatty acids was knocked out, we discovered that these microbial products have immunoglobulin A-modulatory activity.
Antibiotics alter microbiota composition and increase infection susceptibility. However, the generalizable effects of antibiotics on and the contribution of environmental variables to gut commensals remain unclear. To address this, we tracked microbiota dynamics with high temporal and taxonomic resolution during antibiotic treatment in a controlled murine system by isolating variables such as diet, treatment history, and housing co-inhabitants. Human microbiotas were remarkably resilient and recovered during antibiotic treatment, with transient dominance of resistant Bacteroides and taxa-asymmetric diversity reduction. In certain cases, in vitro sensitivities were not predictive of in vivo responses, underscoring the significance of host and community context. A fiber-deficient diet exacerbated microbiota collapse and delayed recovery. Species replacement through cross housing after ciprofloxacin treatment established resilience to a second treatment. Single housing drastically disrupted recovery, highlighting the importance of environmental reservoirs. Our findings highlight deterministic microbiota adaptations to perturbations and the translational potential for modulating diet, sanitation, and microbiota composition during antibiotics.
Gut-derived immunoglobulin A (IgA) is the most abundant antibody secreted in the gut that shapes gut microbiota composition and functionality. However, most of the microbial antigens targeted by gut IgA remain unknown, and the functional effects of IgA targeting these antigens are currently understudied. This study provides a framework for identifying and characterizing gut microbiota antigens targeted by gut IgA. We developed a small intestinal ex vivo culture assay to harvest lamina propria IgA from gnotobiotic mice, with the aim of identifying antigenic targets in a model human gut commensal, Bacteroides thetaiotaomicron VPI-5482. Colonization by B. thetaiotaomicron induced a microbe-specific IgA response that was reactive against diverse antigens, including capsular polysaccharides, lipopolysaccharides, and proteins. IgA against microbial protein antigens targeted membrane and secreted proteins with diverse functionalities, including an IgA specific against proteins of the polysaccharide utilization locus (PUL) that are necessary for utilization of fructan, which is an important dietary polysaccharide. Further analyses demonstrated that the presence of dietary fructan increased the production of fructan PUL-specific IgA, which then downregulated the expression of fructan PUL in B. thetaiotaomicron, both in vivo and in vitro Since the expression of fructan PUL has been associated with the ability of B. thetaiotaomicron to colonize the gut in the presence of dietary fructans, our work suggests a novel role for gut IgA in regulating microbial colonization by modulating their metabolism. IMPORTANCE Given the significant impact that gut microbes have on our health, it is essential to identify key host and environmental factors that shape this diverse community. While many studies have highlighted the impact of diet on gut microbiota, little is known about how the host regulates this critical diet-microbiota interaction. In our present study, we discovered that gut IgA targeted a protein complex involved in the utilization of an important dietary polysaccharide: fructan. While the presence of dietary fructans was previously thought to allow unrestricted growth of fructan-utilizing bacteria, our work shows that gut IgA, by targeting proteins responsible for fructan utilization, provides the host with tools that can restrict the microbial utilization of such polysaccharides, thereby controlling their growth.
The human body is an ecosystem that is home to a complex array of microbes known as the microbiome or microbiota. This ecosystem plays an important role in human health, but as a result of recent lifestyle changes occurring around the planet, whole populations are seeing a major shift in their gut microbiota. Measures meant to kill or limit exposure to pathogenic microbes, such as antibiotics and sanitation, combined with other factors such as processed food, have had unintended consequences for the human microbial ecosystem, including changes that may be difficult to reverse. Microbiota alteration and the accompanying loss of certain functional attributes might result in the microbial communities of people living in industrialized societies being suboptimal for human health. As macroecologists, conservationists, and climate scientists race to document, understand, predict, and delay global changes in our wider environment, microbiota scientists may benefit by using analogous approaches to study and protect our intimate microbial ecosystems.
A perturbed gut microbiome has recently been linked with multiple disease processes, yet researchers currently lack tools that can provide in vivo, quantitative, and real-time insight into these processes and associated host-microbe interactions. We propose an in vivo wireless implant for monitoring gastrointestinal tract redox states using oxidation-reduction potentials (ORP). The implant is powered and conveniently interrogated via ultrasonic waves. We engineer the sensor electronics, electrodes, and encapsulation materials for robustness in vivo, and integrate them into an implant that endures autoclave sterilization and measures ORP for 12 days implanted in the cecum of a live rat. The presented implant platform paves the way for long-term experimental testing of biological hypotheses, offering new opportunities for understanding gut redox pathophysiology mechanisms, and facilitating translation to disease diagnosis and treatment applications.
The gut microbiota is a complex and plastic network of diverse organisms intricately connected with human physiology. Recent advances in profiling approaches of both the microbiota and the immune system now enable a deeper exploration of immunity-microbiota connections. An important next step is to elucidate a human-relevant "map" of microbial-immune wiring while focusing on animal studies to probe a prioritized subset of interactions. Here, we provide an overview of this field's current status and discuss two approaches for establishing priorities for detailed investigation: (1) longitudinal intervention studies in humans probing the dynamics of both the microbiota and the immune system and (2) the study of traditional populations to assess lost features of human microbial identity whose absence may be contributing to the rise of immunological disorders. These human-centered approaches offer a judicious path forward to understand the impact of the microbiota in immune development and function.
A reduction in intestinal barrier function is currently believed to play an important role in pathogenesis of many diseases, as it facilitates passage of injurious factors such as lipopolysaccharide, peptidoglycan, whole bacteria, and other toxins to traverse the barrier to damage the intestine or enter the portal circulation. Currently available evidence in animal models and in vitro systems has shown that certain dietary interventions can be used to reinforce the intestinal barrier to prevent the development of disease. The relevance of these studies to human health is unknown. Herein, we define the components of the intestinal barrier, review available modalities to assess its structure and function in humans, and review the available evidence in model systems or perturbations in humans that diet can be used to fortify intestinal barrier function. Acknowledging the technical challenges and the present gaps in knowledge, we provide a conceptual framework by which evidence could be developed to support the notion that diet can reinforce human intestinal barrier function to restore normal function and potentially reduce the risk for disease. Such evidence would provide information on the development of healthier diets and serve to provide a framework by which federal agencies such as the US Food and Drug Administration can evaluate evidence linking diet with normal human structure/function claims focused on reducing risk of disease in the general public.
Human-associated microbial communities have adapted to environmental pressures. Doses of antibiotics select for a community with increased antibiotic resistance, inflammation is accompanied by expansion of community members equipped to flourish in the presence of immune effectors and Western diets shift the microbiota away from fibre degraders in favour of species that thrive on mucus. Recent data suggest that the microbiota of industrialized societies differs substantially from the recent ancestral microbiota of humans. Rapid modernization, including medical practices and dietary changes, is causing progressive deterioration of the microbiota, and we hypothesize that this may contribute to various diseases prevalent in industrialized societies. In this Opinion article, we explore whether individuals in the industrialized world may be harbouring a microbial community that, while compatible with our environment, is now incompatible with our human biology.
Small intestinal bacterial overgrowth (SIBO) has been implicated in symptoms associated with functional gastrointestinal disorders (FGIDs), though mechanisms remain poorly defined and treatment involves non-specific antibiotics. Here we show that SIBO based on duodenal aspirate culture reflects an overgrowth of anaerobes, does not correspond with patient symptoms, and may be a result of dietary preferences. Small intestinal microbial composition, on the other hand, is significantly altered in symptomatic patients and does not correspond with aspirate culture results. In a pilot interventional study we found that switching from a high fiber diet to a low fiber, high simple sugar diet triggered FGID-related symptoms and decreased small intestinal microbial diversity while increasing small intestinal permeability. Our findings demonstrate that characterizing small intestinal microbiomes in patients with gastrointestinal symptoms may allow a more targeted antibacterial or a diet-based approach to treatment.
Sepsis is a deleterious immune response to infection that leads to organ failure and is the 11th most common cause of death worldwide. Despite plaguing humanity for thousands of years, the host factors that regulate this immunological response and subsequent sepsis severity and outcome are not fully understood. Here we describe how the Western diet (WD), a diet high in fat and sucrose and low in fiber, found rampant in industrialized countries, leads to worse disease and poorer outcomes in an LPS-driven sepsis model in WD-fed mice compared with mice fed standard fiber-rich chow (SC). We find that WD-fed mice have higher baseline inflammation (metaflammation) and signs of sepsis-associated immunoparalysis compared with SC-fed mice. WD mice also have an increased frequency of neutrophils, some with an "aged" phenotype, in the blood during sepsis compared with SC mice. Importantly, we found that the WD-dependent increase in sepsis severity and higher mortality is independent of the microbiome, suggesting that the diet may be directly regulating the innate immune system through an unknown mechanism. Strikingly, we could predict LPS-driven sepsis outcome by tracking specific WD-dependent disease factors (e.g., hypothermia and frequency of neutrophils in the blood) during disease progression and recovery. We conclude that the WD is reprogramming the basal immune status and acute response to LPS-driven sepsis and that this correlates with alternative disease paths that lead to more severe disease and poorer outcomes.
The composition of the gut microbiome in industrialized populations differs from those living traditional lifestyles. However, it has been difficult to separate the contributions of human genetic and geographic factors from lifestyle. Whether shifts away from the foraging lifestyle that characterize much of humanity's past influence the gut microbiome, and to what degree, remains unclear. Here, we characterize the stool bacterial composition of four Himalayan populations to investigate how the gut community changes in response to shifts in traditional human lifestyles. These groups led seminomadic hunting-gathering lifestyles until transitioning to varying levels of agricultural dependence upon farming. The Tharu began farming 250-300 years ago, the Raute and Raji transitioned 30-40 years ago, and the Chepang retain many aspects of a foraging lifestyle. We assess the contributions of dietary and environmental factors on their gut-associated microbes and find that differences in the lifestyles of Himalayan foragers and farmers are strongly correlated with microbial community variation. Furthermore, the gut microbiomes of all four traditional Himalayan populations are distinct from that of the Americans, indicating that industrialization may further exacerbate differences in the gut community. The Chepang foragers harbor an elevated abundance of taxa associated with foragers around the world. Conversely, the gut microbiomes of the populations that have transitioned to farming are more similar to those of Americans, with agricultural dependence and several associated lifestyle and environmental factors correlating with the extent of microbiome divergence from the foraging population. The gut microbiomes of Raute and Raji reveal an intermediate state between the Chepang and Tharu, indicating that divergence from a stereotypical foraging microbiome can occur within a single generation. Our results also show that environmental factors such as drinking water source and solid cooking fuel are significantly associated with the gut microbiome. Despite the pronounced differences in gut bacterial composition across populations, we found little differences in alpha diversity across lifestyles. These findings in genetically similar populations living in the same geographical region establish the key role of lifestyle in determining human gut microbiome composition and point to the next challenging steps of determining how large-scale gut microbiome reconfiguration impacts human biology.
While the gut microbiota's malleability makes it highly responsive to our environment, it also renders it susceptible to rapid selection by factors associated with an industrialized lifestyle. Recently, in Cell, Vangay et al. (2018) have tracked and revealed rapid and profound changes in the gut community of immigrants to one that resembles long-term residents of the U.S.
The gut microbiota plays a critical role in pathogen defense. Studies using antibiotic-treated mice reveal mechanisms that increase susceptibility to Clostridioides difficile infection (CDI), but risk factors associated with CDI in humans extend beyond antibiotic use. Here, we studied the dysbiotic gut microbiota of a subset of patients with diarrhea and modeled the gut microbiota of these patients by fecal transplantation into germ-free mice. When challenged with C. difficile, the germ-free mice transplanted with fecal samples from patients with dysbiotic microbial communities showed increased gut amino acid concentrations and greater susceptibility to CDI. A C. difficile mutant that was unable to use proline as an energy source was unable to robustly infect germ-free mice transplanted with a dysbiotic or healthy human gut microbiota. Prophylactic dietary intervention using a low-proline or low-protein diet in germ-free mice colonized by a dysbiotic human gut microbiota resulted in decreased expansion of wild-type C. difficile after challenge, suggesting that amino acid availability might be important for CDI. Furthermore, a prophylactic fecal microbiota transplant in mice with dysbiosis reduced proline availability and protected the mice from CDI. Last, we identified clinical risk factors that could potentially predict gut microbial dysbiosis and thus greater susceptibility to CDI in a retrospective cohort of patients with diarrhea. Identifying at-risk individuals and reducing their susceptibility to CDI through gut microbiota-targeted therapies could be a new approach to preventing C. difficile infection in susceptible patients.
A 2-day workshop organized by the National Institutes of Health and U.S. Department of Agriculture included 16 presentations focused on the role of diet in alterations of the gastrointestinal microbiome, primarily that of the colon. Although thousands of research projects have been funded by U.S. federal agencies to study the intestinal microbiome of humans and a variety of animal models, only a minority addresses dietary effects, and a small subset is described in sufficient detail to allow reproduction of a study. Whereas there are standards being developed for many aspects of microbiome studies, such as sample collection, nucleic acid extraction, data handling, etc., none has been proposed for the dietary component; thus this workshop focused on the latter specific point. It is important to foster rigor in design and reproducibility of published studies to maintain high quality and enable designs that can be compared in systematic reviews. Speakers addressed the influence of the structure of the fermentable carbohydrate on the microbiota and the variables to consider in design of studies using animals, in vitro models, and human subjects. For all types of studies, strengths and weaknesses of various designs were highlighted, and for human studies, comparisons between controlled feeding and observational designs were discussed. Because of the lack of published, best-diet formulations for specific research questions, the main recommendation is to describe dietary ingredients and treatments in as much detail as possible to allow reproduction by other scientists.
The study of traditional populations provides a view of human-associated microbes unperturbed by industrialization, as well as a window into the microbiota that co-evolved with humans. Here we discuss our recent work characterizing the microbiota from the Hadza hunter-gatherers of Tanzania. We found seasonal shifts in bacterial taxa, diversity, and carbohydrate utilization by the microbiota. When compared to the microbiota composition from other populations around the world, the Hadza microbiota shares bacterial families with other traditional societies that are rare or absent from microbiotas of industrialized nations. We present additional observations from the Hadza microbiota and their lifestyle and environment, including microbes detected on hands, water, and animal sources, how the microbiota varies with sex and age, and the short-term effects of introducing agricultural products into the diet. In the context of our previously published findings and of these additional observations, we discuss a path forward for future work.
The intestinal microbiota provides colonization resistance against pathogens, limiting pathogen expansion and transmission. These microbiota-mediated mechanisms were previously identified by observing loss of colonization resistance after antibiotic treatment or dietary changes, which severely disrupt microbiota communities. We identify a microbiota-mediated mechanism of colonization resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) by comparing high-complexity commensal communities with different levels of colonization resistance. Using inbred mouse strains with different infection dynamics and S. Typhimurium intestinal burdens, we demonstrate that Bacteroides species mediate colonization resistance against S. Typhimurium by producing the short-chain fatty acid propionate. Propionate directly inhibits pathogen growth in vitro by disrupting intracellular pH homeostasis, and chemically increasing intestinal propionate levels protects mice from S. Typhimurium. In addition, administering susceptible mice Bacteroides, but not a propionate-production mutant, confers resistance to S. Typhimurium. This work provides mechanistic understanding into the role of individualized microbial communities in host-to-host variability of pathogen transmission.
Osmotic diarrhea is a prevalent condition in humans caused by food intolerance, malabsorption, and widespread laxative use. Here, we assess the resilience of the gut ecosystem to osmotic perturbation at multiple length and timescales using mice as model hosts. Osmotic stress caused reproducible extinction of highly abundant taxa and expansion of less prevalent members in human and mouse microbiotas. Quantitative imaging revealed decimation of the mucus barrier during osmotic perturbation, followed by recovery. The immune system exhibited temporary changes in cytokine levels and a lasting IgG response against commensal bacteria. Increased osmolality prevented growth of commensal strains in vitro, revealing one mechanism contributing to extinction. Environmental availability of microbiota members mitigated extinction events, demonstrating how species reintroduction can affect community resilience. Our findings (1) demonstrate that even mild osmotic diarrhea can cause lasting changes to the microbiota and host and (2) lay the foundation for interventions that increase system-wide resilience.
Tryptamine, a tryptophan-derived monoamine similar to 5-hydroxytryptamine (5-HT), is produced by gut bacteria and is abundant in human and rodent feces. However, the physiologic effect of tryptamine in the gastrointestinal (GI) tract remains unknown. Here, we show that the biological effects of tryptamine are mediated through the 5-HT4 receptor (5-HT4R), a G-protein-coupled receptor (GPCR) uniquely expressed in the colonic epithelium. Tryptamine increases both ionic flux across the colonic epithelium and fluid secretion in colonoids from germ-free (GF) and humanized (ex-GF colonized with human stool) mice, consistent with increased intestinal secretion. The secretory effect of tryptamine is dependent on 5-HT4R activation and is blocked by 5-HT4R antagonist and absent in 5-HT4R-/- mice. GF mice colonized by Bacteroides thetaiotaomicron engineered to produce tryptamine exhibit accelerated GI transit. Our study demonstrates an aspect of host physiology under control of a bacterial metabolite that can be exploited as a therapeutic modality.
Genomic differences between gut-resident bacterial strains likely underlie significant interindividual variation in microbiome function. Traditional methods of determining community composition, such as 16S rRNA gene amplicon sequencing, fail to capture this functional diversity. Metagenomic approaches are a significant step forward in identifying strain-level sequence variants; however, given the current paucity of biochemical information, they too are limited to mainly low-resolution and incomplete functional predictions. Using genomic, biochemical, and molecular approaches, we identified differences in the fructan utilization profiles of two closely related Bacteroides thetaiotaomicron strains. B. thetaiotaomicron 8736 (Bt-8736) contains a fructan polysaccharide utilization locus (PUL) with a divergent susC/susD homolog gene pair that enables it to utilize inulin, differentiating this strain from other characterized Bt strains. Transfer of the distinct pair of susC/susD genes from Bt-8736 into the noninulin using type strain B. thetaiotaomicronVPI-5482 resulted in inulin use by the recipient strain, Bt(8736-2). The presence of the divergent susC/susD gene pair alone enabled the hybrid Bt(8736-2) strain to outcompete the wild-type strain in vivo in mice fed an inulin diet. Further, we discovered that the susC/susD homolog gene pair facilitated import of inulin into the periplasm without surface predigestion by an endo-acting enzyme, possibly due to the short average chain length of inulin compared to many other polysaccharides. Our data builds upon recent reports of dietary polysaccharide utilization mechanisms found in members of the Bacteroides genus and demonstrates how the acquisition of two genes can alter the functionality and success of a strain within the gut. IMPORTANCE Dietary polysaccharides play a dominant role in shaping the composition and functionality of our gut microbiota. Dietary interventions using these microbiota-accessible carbohydrates (MACs) serve as a promising tool for manipulating the gut microbial community. However, our current gap in knowledge regarding microbial metabolic pathways that are involved in the degradation of these MACs has made the design of rational interventions difficult. The issue is further complicated by the diversity of pathways observed for the utilization of similar MACs, even in closely related microbial strains. Our current work focuses on divergent fructan utilization pathways in two closely related B. thetaiotaomicron strains and provides an integrated approach to characterize the molecular basis for strain-level functional differences.
The dense microbial ecosystem in the gut is intimately connected to numerous facets of human biology, and manipulation of the gut microbiota has broad implications for human health. In the absence of profound perturbation, the bacterial strains that reside within an individual are mostly stable over time. By contrast, the fate of exogenous commensal and probiotic strains applied to an established microbiota is variable, generally unpredictable and greatly influenced by the background microbiota. Therefore, analysis of the factors that govern strain engraftment and abundance is of critical importance to the emerging field of microbiome reprogramming. Here we generate an exclusive metabolic niche in mice via administration of a marine polysaccharide, porphyran, and an exogenous Bacteroides strain harbouring a rare gene cluster for porphyran utilization. Privileged nutrient access enables reliable engraftment of the exogenous strain at predictable abundances in mice harbouring diverse communities of gut microbes. This targeted dietary support is sufficient to overcome priority exclusion by an isogenic strain, and enables strain replacement. We demonstrate transfer of the 60-kb porphyran utilization locus into a naive strain of Bacteroides, and show finely tuned control of strain abundance in the mouse gut across multiple orders of magnitude by varying porphyran dosage. Finally, we show that this system enables the introduction of a new strain into the colonic crypt ecosystem. These data highlight the influence of nutrient availability in shaping microbiota membership, expand the ability to perform a broad spectrum of investigations in the context of a complex microbiota, and have implications for cell-based therapeutic strategies in the gut.
Clostridium difficile is an opportunistic diarrhoeal pathogen, and C. difficile infection (CDI) represents a major health care concern, causing an estimated 15,000 deaths per year in the United States alone. Several enteric pathogens, including C. difficile, leverage inflammation and the accompanying microbial dysbiosis to thrive in the distal gut. Although diet is among the most powerful available tools for affecting the health of humans and their relationship with their microbiota, investigation into the effects of diet on CDI has been limited. Here, we show in mice that the consumption of microbiota-accessible carbohydrates (MACs) found in dietary plant polysaccharides has a significant effect on CDI. Specifically, using a model of antibiotic-induced CDI that typically resolves within 12 days of infection, we demonstrate that MAC-deficient diets perpetuate CDI. We show that C. difficile burdens are suppressed through the addition of either a diet containing a complex mixture of MACs or a simplified diet containing inulin as the sole MAC source. We show that switches between these dietary conditions are coincident with changes to microbiota membership, its metabolic output and C. difficile-mediated inflammation. Together, our data demonstrate the outgrowth of MAC-utilizing taxa and the associated end products of MAC metabolism, namely, the short-chain fatty acids acetate, propionate and butyrate, are associated with decreased C. difficile fitness despite increased C. difficile toxin expression in the gut. Our findings, when placed into the context of the known fibre deficiencies of a human Western diet, provide rationale for pursuing MAC-centric dietary strategies as an alternate line of investigation for mitigating CDI.
In this prospective cohort study, we examine the feasibility of a protocol to optimize microbiota for fecal microbiota transplantation (FMT). Donor stool metrics generally accepted as markers of gut health were used to select a stool donor based on superior microbial diversity, balanced constitution of Bacteroidetes versus Firmicutes and high concentration of fecal butyrate. Selected donor microbiota was then administered via FMT. A total of 10 patients with median age of 12 years with recurrent Clostridium difficile infection received the intervention. The rate of recurrence-free resolution with 1-2 FMTs was 100% at Week 10. With a single FMT, 80% of patients cleared Clostridium difficile infection without recurrence, whereas 20% of patients required a single re-treatment. No serious adverse events occurred. Microbiota sequencing revealed that recipients' gut microbiota phylogenic diversity increased by 72-hours post-transplantation, with sustainment over 10-week follow-up. This study highlights the feasibility of purposefully selecting the most ideal microbiota for transplantation.
The development of experimental techniques capable of probing the viscoelasticity of soft materials over a broad range of time scales is essential to uncovering the physics that governs their behavior. In this work, we develop a microrheology technique that requires only 12 μL of sample and is capable of resolving dynamic behavior ranging in time scales from 10-6 to 10 s. Our approach, based on dynamic light scattering in the single-scattering limit, enables the study of polymer gels and other soft materials over a vastly larger hierarchy of time scales than macrorheology measurements. Our technique captures the viscoelastic modulus of polymer hydrogels with a broad range of stiffnesses from 10 to 104 Pa. We harness these capabilities to capture hierarchical molecular relaxations in DNA and to study the rheology of precious biological materials that are impractical for macrorheology measurements, including decellularized extracellular matrices and intestinal mucus. The use of a commercially available benchtop setup that is already available to a variety of soft matter researchers renders microrheology measurements accessible to a broader range of users than existing techniques, with the potential to reveal the physics that underlies complex polymer hydrogels and biological materials.
The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream, where they can have systemic effects on the host. Although these small molecules commonly reach concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes, we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community.
Although humans have cospeciated with their gut-resident microbes, it is difficult to infer features of our ancestral microbiome. Here, we examine the microbiome profile of 350 stool samples collected longitudinally for more than a year from the Hadza hunter-gatherers of Tanzania. The data reveal annual cyclic reconfiguration of the microbiome, in which some taxa become undetectable only to reappear in a subsequent season. Comparison of the Hadza data set with data collected from 18 populations in 16 countries with varying lifestyles reveals that gut community membership corresponds to modernization: Notably, the taxa within the Hadza that are the most seasonally volatile similarly differentiate industrialized and traditional populations. These data indicate that some dynamic lineages of microbes have decreased in prevalence and abundance in modernized populations.
Applying synthetic biology to engineer gut-resident microbes provides new avenues to investigate microbe-host interactions, perform diagnostics, and deliver therapeutics. Here, we describe a platform for engineering Bacteroides, the most abundant genus in the Western microbiota, which includes a process for high-throughput strain modification. We have identified a novel phage promoter and translational tuning strategy and achieved an unprecedented level of expression that enables imaging of fluorescent-protein-expressing Bacteroides stably colonizing the mouse gut. A detailed characterization of the phage promoter has provided a set of constitutive promoters that span over four logs of strength without detectable fitness burden within the gut over 14 days. These promoters function predictably over a 1,000,000-fold expression range in phylogenetically diverse Bacteroides species. With these promoters, unique fluorescent signatures were encoded to allow differentiation of six species within the gut. Fluorescent protein-based differentiation of isogenic strains revealed that priority of gut colonization determines colonic crypt occupancy.
The first rudimentary evidence that the human body harbors a microbiota hinted at the complexity of host-associated microbial ecosystems. Now, almost 400 years later, a renaissance in the study of microbiota spatial organization, driven by coincident revolutions in imaging and sequencing technologies, is revealing functional relationships between biogeography and health, particularly in the vertebrate gut. In this Review, we present our current understanding of principles governing the localization of intestinal bacteria, and spatial relationships between bacteria and their hosts. We further discuss important emerging directions that will enable progressing from the inherently descriptive nature of localization and -omics technologies to provide functional, quantitative, and mechanistic insight into this complex ecosystem.
Regulatory T cells (Tregs) are required to establish immune tolerance to commensal microbes. Tregs accumulate abruptly in the skin during a defined window of postnatal tissue development. However, the mechanisms mediating Treg migration to neonatal skin are unknown. Here we show that hair follicle (HF) development facilitates the accumulation of Tregs in neonatal skin and that upon skin entry these cells localize to HFs, a primary reservoir for skin commensals. Further, germ-free neonates had reduced skin Tregs indicating that commensal microbes augment Treg accumulation. We identified Ccl20 as a HF-derived, microbiota-dependent chemokine and found its receptor, Ccr6, to be preferentially expressed by Tregs in neonatal skin. The Ccl20-Ccr6 pathway mediated Treg migration in vitro and in vivo. Thus, HF morphogenesis, commensal microbe colonization, and local chemokine production work in concert to recruit Tregs into neonatal skin, thereby establishing this tissue Treg niche early in life.
It is widely accepted that Clostridium difficile exploits dysbiosis and leverages inflammation to thrive in the gut environment, where it can asymptomatically colonize humans or cause a toxin-mediated disease ranging in severity from frequent watery diarrhea to pseudomembranous colitis or toxic megacolon. Here, we synthesize recent findings from the gut microbiota and enteric pathogenesis fields to inform the next steps toward a better understanding of C. difficile infection (CDI). In this review, we present a model in which the lifestyle of C. difficile is dictated by the metabolic state of the distal gut ecosystem. Contributions by C. difficile (specifically the production and action of the large glycosylating toxins TcdA and TcdB), the microbiota, and the host dictate whether the gut environment is supportive to the pathogen. Mechanistic, metabolic pathway-focused approaches encompassing the roles of all of these players are helping to elucidate the molecular ecology of the distal gut underlying a diseased or healthy ecosystem. A new generation of therapeutic strategies that are more targeted (and palatable) than fecal microbiota transplants or broad-spectrum antibiotics will be fueled by insight into the interspecies (host-microbe and microbe-microbe) interactions that differentiate healthy from pathogen-infested microbiotas.
Renal disease is growing in prevalence and has striking co-morbidities with metabolic and cardiovascular disease. Indoxyl sulfate (IS) is a toxin that accumulates in plasma when kidney function declines and contributes to the progression of chronic kidney disease. IS derives exclusively from the gut microbiota. Bacterial tryptophanases convert tryptophan to indole, which is absorbed and modified by the host to produce IS. Here, we identify a widely distributed family of tryptophanases in the gut commensal Bacteroides and find that deleting this gene eliminates the production of indole in vitro. By altering the status or abundance of the Bacteroides tryptophanase, we can modulate IS levels in gnotobiotic mice and in the background of a conventional murine gut community. Our results demonstrate that it is possible to control host IS levels by targeting the microbiota and suggest a possible strategy for treating renal disease.
Diet plays an important role in shaping the structure and function of the gut microbiota. The microbes and microbial products in turn can influence various aspects of host physiology. One promising route to affect host function and restore health is by altering the gut microbiome using dietary intervention. The individuality of the microbiome may pose a significant challenge, so we sought to determine how different microbiotas respond to the same dietary intervention in a controlled setting. We modeled gut microbiotas from three healthy donors in germfree mice and defined compositional and functional alteration following a change in dietary microbiota-accessible carbohydrates (MACs). The three gut communities exhibited responses that differed markedly in magnitude and in the composition of microbiota-derived metabolites. Adjustments in community membership did not correspond to the magnitude of changes in the microbial metabolites, highlighting potential challenges in predicting functional responses from compositional data and the need to assess multiple microbiota parameters following dietary interventions. IMPORTANCE Dietary modification has long been used empirically to modify symptoms in inflammatory bowel disease, irritable bowel syndrome, and a diverse group of diseases with gastrointestinal symptoms. There is both anecdotal and scientific evidence to suggest that individuals respond quite differently to similar dietary changes, and the highly individualized nature of the gut microbiota makes it a prime candidate for these differences. To overcome the typical confounding factors of human dietary interventions, here we employ ex-germfree mice colonized by microbiotas of three different humans to test how different microbiotas respond to a defined change in carbohydrate content of diet by measuring changes in microbiota composition and function using marker gene-based next-generation sequencing and metabolomics. Our findings suggest that the same diet has very different effects on each microbiota's membership and function, which may in turn explain interindividual differences in response to a dietary ingredient.
It is widely accepted that obesity and associated metabolic diseases, including type 2 diabetes, are intimately linked to diet. However, the gut microbiota has also become a focus for research at the intersection of diet and metabolic health. Mechanisms that link the gut microbiota with obesity are coming to light through a powerful combination of translation-focused animal models and studies in humans. A body of knowledge is accumulating that points to the gut microbiota as a mediator of dietary impact on the host metabolic status. Efforts are focusing on the establishment of causal relationships in people and the prospect of therapeutic interventions such as personalized nutrition.
Improved understanding of the interplay between host and microbes stands to illuminate new avenues for disease diagnosis, treatment, and prevention. Here, we provide a high-resolution view of the dynamics between host and gut microbiota during antibiotic-induced intestinal microbiota depletion, opportunistic Salmonella typhimurium and Clostridium difficile pathogenesis, and recovery from these perturbed states in a mouse model. Host-centric proteome and microbial community profiles provide a nuanced longitudinal view, revealing the interdependence between host and microbiota in evolving dysbioses. Time- and condition-specific molecular and microbial signatures are evident and clearly distinguished from pathogen-independent inflammatory fingerprints. Our data reveal that mice recovering from antibiotic treatment or C. difficile infection retain lingering signatures of inflammation, despite compositional normalization of the microbiota, and host responses could be rapidly and durably relieved through fecal transplant. These experiments demonstrate insights that emerge from the combination of these orthogonal, untargeted approaches to the gastrointestinal ecosystem.
The gut is home to trillions of microorganisms that have fundamental roles in many aspects of human biology, including immune function and metabolism. The reduced diversity of the gut microbiota in Western populations compared to that in populations living traditional lifestyles presents the question of which factors have driven microbiota change during modernization. Microbiota-accessible carbohydrates (MACs) found in dietary fibre have a crucial involvement in shaping this microbial ecosystem, and are notably reduced in the Western diet (high in fat and simple carbohydrates, low in fibre) compared with a more traditional diet. Here we show that changes in the microbiota of mice consuming a low-MAC diet and harbouring a human microbiota are largely reversible within a single generation. However, over several generations, a low-MAC diet results in a progressive loss of diversity, which is not recoverable after the reintroduction of dietary MACs. To restore the microbiota to its original state requires the administration of missing taxa in combination with dietary MAC consumption. Our data illustrate that taxa driven to low abundance when dietary MACs are scarce are inefficiently transferred to the next generation, and are at increased risk of becoming extinct within an isolated population. As more diseases are linked to the Western microbiota and the microbiota is targeted therapeutically, microbiota reprogramming may need to involve strategies that incorporate dietary MACs as well as taxa not currently present in the Western gut.
Use of sophisticated reductionist and whole-system approaches are providing much-needed technologies to unravel the complex mélange of microbiome functions.
Endogenous intestinal microbiota have wide-ranging and largely uncharacterized effects on host physiology. Here, we used reverse-phase liquid chromatography-coupled tandem mass spectrometry to define the mouse intestinal proteome in the stomach, jejunum, ileum, cecum and proximal colon under three colonization states: germ-free (GF), monocolonized with Bacteroides thetaiotaomicron and conventionally raised (CR). Our analysis revealed distinct proteomic abundance profiles along the gastrointestinal (GI) tract. Unsupervised clustering showed that host protein abundance primarily depended on GI location rather than colonization state and specific proteins and functions that defined these locations were identified by random forest classifications. K-means clustering of protein abundance across locations revealed substantial differences in host protein production between CR mice relative to GF and monocolonized mice. Finally, comparison with fecal proteomic data sets suggested that the identities of stool proteins are not biased to any region of the GI tract, but are substantially impacted by the microbiota in the distal colon.
Genomic technologies have significantly advanced our understanding of the composition and diversity of host-associated microbial populations. However, their spatial organization and functional interactions relative to the host have been more challenging to study. Here we present a pipeline for the assessment of intestinal microbiota localization within immunofluorescence images of fixed gut cross-sections that includes a flexible software package, BacSpace, for high-throughput quantification of microbial organization. Applying this pipeline to gnotobiotic and human microbiota-colonized mice, we demonstrate that elimination of microbiota-accessible carbohydrates (MACs) from the diet results in thinner mucus in the distal colon, increased proximity of microbes to the epithelium, and heightened expression of the inflammatory marker REG3β. Measurements of microbe-microbe proximity reveal that a MAC-deficient diet alters monophyletic spatial clustering. Furthermore, we quantify the invasion of Helicobacter pylori into the glands of the mouse stomach relative to host mitotic progenitor cells, illustrating the generalizability of this approach.
Clostridium difficile infection (CDI) is a worldwide health threat that is typically triggered by the use of broad-spectrum antibiotics, which disrupt the natural gut microbiota and allow this Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of disease coupled with decreased response, high recurrence rates, and emergence of multiple antibiotic-resistant strains have created an urgent need for new therapies. We describe pharmacological targeting of the cysteine protease domain (CPD) within the C. difficile major virulence factor toxin B (TcdB). Through a targeted screen with an activity-based probe for this protease domain, we identified a number of potent CPD inhibitors, including one bioactive compound, ebselen, which is currently in human clinical trials for a clinically unrelated indication. This drug showed activity against both major virulence factors, TcdA and TcdB, in biochemical and cell-based studies. Treatment in a mouse model of CDI that closely resembles the human infection confirmed a therapeutic benefit in the form of reduced disease pathology in host tissues that correlated with inhibition of the release of the toxic glucosyltransferase domain (GTD). Our results show that this non-antibiotic drug can modulate the pathology of disease and therefore could potentially be developed as a therapeutic for the treatment of CDI.
The microbiome has been implicated directly in host health, especially host metabolic processes and development of immune responses. These are particularly important in infants where the gut first begins being colonized, and such processes may be modeled in mice. In this investigation we follow longitudinally the urine metabolome of ex-germ-free mice, which are colonized with two bacterial species, Bacteroides thetaiotaomicron and Bifidobacterium longum. High-throughput mass spectrometry profiling of urine samples revealed dynamic changes in the metabolome makeup, associated with the gut bacterial colonization, enabled by our adaptation of non-linear time-series analysis to urine metabolomics data. Results demonstrate both gradual and punctuated changes in metabolite production and that early colonization events profoundly impact the nature of small molecules circulating in the host. The identified small molecules are implicated in amino acid and carbohydrate metabolic processes, and offer insights into the dynamic changes occurring during the colonization process, using high-throughput longitudinal methodology.
The gastrointestinal (GI) ecosystem is increasingly understood to be a fundamental component of health, and has been identified as a new focal point for diagnosing, correcting and preventing countless disorders. Shotgun DNA sequencing has emerged as the dominant technology for determining the genetic and microbial composition of the gut microbiota. This technology has linked microbiota dysbioses to numerous GI diseases including inflammatory bowel disease, obesity and allergy, and to non-GI diseases like autism and depression. The importance of establishing causality in the deterioration of the host-microbiota relationship is well appreciated; however, discovery of candidate molecules and pathways that underlie mechanisms remains a major challenge. Targeted approaches, transcriptional assays, cytokine panels and imaging analyses, applied to animals, have yielded important insight into host responses to the microbiota. However, non-invasive, hypothesis-independent means of measuring host responses in humans are necessary to keep pace with similarly unbiased sequencing efforts that monitor microbes. Mass spectrometry-based proteomics has served this purpose in many other fields, but stool proteins exist in such diversity and dynamic range as to overwhelm conventional proteomics technologies. Focused analysis of host protein secretion into the gut lumen and monitoring proteome-level dynamics in stool provides a tractable route toward non-invasively evaluating dietary, microbial, surgical or pharmacological intervention efficacies. This review is intended to guide GI biologists and clinicians through the methods currently used to elucidate host responses in the gut, with a specific focus on mass spectrometry-based shotgun proteomics applied to the study of host protein dynamics within the GI ecosystem.
Synthetic biology may lead to the creation of smart microbes that can detect and treat disease
Gut microbiota alterations have been described in several diseases with altered gastrointestinal (GI) motility, and awareness is increasing regarding the role of the gut microbiome in modulating GI function. Serotonin [5-hydroxytryptamine (5-HT)] is a key regulator of GI motility and secretion. To determine the relationship among gut microbes, colonic contractility, and host serotonergic gene expression, we evaluated mice that were germ-free (GF) or humanized (HM; ex-GF colonized with human gut microbiota). 5-HT reduced contractile duration in both GF and HM colons. Microbiota from HM and conventionally raised (CR) mice significantly increased colonic mRNAs Tph1 [(tryptophan hydroxylase) 1, rate limiting for mucosal 5-HT synthesis; P < 0.01] and chromogranin A (neuroendocrine secretion; P < 0.01), with no effect on monoamine oxidase A (serotonin catabolism), serotonin receptor 5-HT4, or mouse serotonin transporter. HM and CR mice also had increased colonic Tph1 protein (P < 0.05) and 5-HT concentrations (GF, 17 ± 3 ng/mg; HM, 25 ± 2 ng/mg; and CR, 35 ± 3 ng/mg; P < 0.05). Enterochromaffin (EC) cell numbers (cells producing 5-HT) were unchanged. Short-chain fatty acids (SCFAs) promoted TPH1 transcription in BON cells (human EC cell model). Thus, gut microbiota acting through SCFAs are important determinants of enteric 5-HT production and homeostasis.
Clostridium difficile is a leading cause of antibiotic-associated diarrhea. The mechanisms underlying C. difficile expansion after microbiota disturbance are just emerging. We assessed the gene expression profile of C. difficile within the intestine of gnotobiotic mice to identify genes regulated in response to either dietary or microbiota compositional changes. In the presence of the gut symbiont Bacteroides thetaiotaomicron, C. difficile induces a pathway that metabolizes the microbiota fermentation end-product succinate to butyrate. The low concentration of succinate present in the microbiota of conventional mice is transiently elevated upon antibiotic treatment or chemically induced intestinal motility disturbance, and C. difficile exploits this succinate spike to expand in the perturbed intestine. A C. difficile mutant compromised in succinate utilization is at a competitive disadvantage during these perturbations. Understanding the metabolic mechanisms involved in microbiota-C. difficile interactions may help to identify approaches for the treatment and prevention of C. difficile-associated diseases.
The gut microbiota of a healthy person may not be equivalent to a healthy microbiota. It is possible that the Western microbiota is actually dysbiotic and predisposes individuals to a variety of diseases. The asymmetric plasticity between the relatively stable human genome and the more malleable gut microbiome suggests that incompatibilities between the two could rapidly arise. The Western lifestyle, which includes a diet low in microbiota-accessible carbohydrates (MACs), has selected for a microbiota with altered membership and functionality compared to those of groups living traditional lifestyles. Interactions between resident microbes and host leading to immune dysregulation may explain several diseases that share inflammation as a common basis. The low-MAC Western diet results in poor production of gut microbiota-generated short-chain fatty acids (SCFAs), which attenuate inflammation through a variety of mechanisms in mouse models. Studies focused on modern and traditional societies, combined with animal models, are needed to characterize the connection between diet, microbiota composition, and function. Differentiating between an optimal microbiota, one that increases disease risk, and one that is causative or potentiates disease will be required to further understand both the etiology and possible treatments for health problems related to microbiota dysbiosis.
Fucosyltransferase 2 (FUT2) is an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. The H antigen is an oligosaccharide moiety that acts as both an attachment site and carbon source for intestinal bacteria. Non-secretors, who are homozygous for the loss-of-function alleles of FUT2 gene (sese), have increased susceptibility to Crohn's disease (CD). To characterize the effect of FUT2 polymorphism on the mucosal ecosystem, we profiled the microbiome, meta-proteome and meta-metabolome of 75 endoscopic lavage samples from the cecum and sigmoid of 39 healthy subjects (12 SeSe, 18 Sese and 9 sese). Imputed metagenomic analysis revealed perturbations of energy metabolism in the microbiome of non-secretor and heterozygote individuals, notably the enrichment of carbohydrate and lipid metabolism, cofactor and vitamin metabolism and glycan biosynthesis and metabolism-related pathways, and the depletion of amino-acid biosynthesis and metabolism. Similar changes were observed in mice bearing the FUT2(-/-) genotype. Metabolomic analysis of human specimens revealed concordant as well as novel changes in the levels of several metabolites. Human metaproteomic analysis indicated that these functional changes were accompanied by sub-clinical levels of inflammation in the local intestinal mucosa. Therefore, the colonic microbiota of non-secretors is altered at both the compositional and functional levels, affecting the host mucosal state and potentially explaining the association of FUT2 genotype and CD susceptibility.
The gut microbiota is a dense and diverse microbial community governed by dynamic microbe-microbe and microbe-host interactions, the status of which influences whether enteric pathogens can cause disease. Here we review recent insights into the key roles that nutrients play in bacterial pathogen exploitation of the gut microbial ecosystem. We synthesize recent findings to support a five-stage model describing the transition between a healthy microbiota and one dominated by a pathogen and disease. Within this five-stage model, two stages are critical to the pathogen: (i) an initial expansion phase that must occur in the absence of pathogen-induced inflammation, followed by (ii) pathogen-promoting physiological changes such as inflammation and diarrhoea. We discuss how this emerging paradigm of pathogen life within the lumen of the gut is giving rise to novel therapeutic strategies.
Consistent compositional shifts in the gut microbiota are observed in IBD and other chronic intestinal disorders and may contribute to pathogenesis. The identities of microbial biomolecular mechanisms and metabolic products responsible for disease phenotypes remain to be determined, as do the means by which such microbial functions may be therapeutically modified. The composition of the microbiota and metabolites in gut microbiome samples in 47 subjects were determined. Samples were obtained by endoscopic mucosal lavage from the cecum and sigmoid colon regions, and each sample was sequenced using the 16S rRNA gene V4 region (Illumina-HiSeq 2000 platform) and assessed by UPLC mass spectroscopy. Spearman correlations were used to identify widespread, statistically significant microbial-metabolite relationships. Metagenomes for identified microbial OTUs were imputed using PICRUSt, and KEGG metabolic pathway modules for imputed genes were assigned using HUMAnN. The resulting metabolic pathway abundances were mostly concordant with metabolite data. Analysis of the metabolome-driven distribution of OTU phylogeny and function revealed clusters of clades that were both metabolically and metagenomically similar. The results suggest that microbes are syntropic with mucosal metabolome composition and therefore may be the source of and/or dependent upon gut epithelial metabolites. The consistent relationship between inferred metagenomic function and assayed metabolites suggests that metagenomic composition is predictive to a reasonable degree of microbial community metabolite pools. The finding that certain metabolites strongly correlate with microbial community structure raises the possibility of targeting metabolites for monitoring and/or therapeutically manipulating microbial community function in IBD and other chronic diseases.
The dense microbial ecosystem within the gut is connected through a complex web of metabolic interactions. In this issue of Cell Host & Microbe, Degnan et al. (2014) establish the importance of different vitamin B12 transporters that help a Bacteroides species acquire vitamins from the environment to maintain a competitive edge.
Siglecs are sialic acid-binding Ig-like lectins that recognize sialoglycans via amino-terminal V-set domains. CD33-related Siglecs (CD33rSiglecs) on innate immune cells recognize endogenous sialoglycans as "self-associated molecular patterns" (SAMPs), dampening immune responses via cytosolic immunoreceptor tyrosine-based inhibition motifs that recruit tyrosine phosphatases. However, sialic acid-expressing pathogens subvert this mechanism through molecular mimicry. Meanwhile, endogenous host SAMPs must continually evolve to evade other pathogens that exploit sialic acids as invasion targets. We hypothesized that these opposing selection forces have accelerated CD33rSiglec evolution. We address this by comparative analysis of major CD33rSiglec (Siglec-3, Siglec-5, and Siglec-9) orthologs in humans, chimpanzees, and baboons. Recombinant soluble molecules displaying ligand-binding domains show marked quantitative and qualitative interspecies differences in interactions with strains of the sialylated pathogen, group B Streptococcus, and with sialoglycans presented as gangliosides or in the form of sialoglycan microarrays, including variations such as N-glycolyl and O-acetyl groups. Primate Siglecs also show quantitative and qualitative intra- and interspecies variations in expression patterns on leukocytes, both in circulation and in tissues. Taken together our data explain why the CD33rSiglec-encoding gene cluster is undergoing rapid evolution via multiple mechanisms, driven by the need to maintain self-recognition by innate immune cells, while escaping 2 distinct mechanisms of pathogen subversion.
We investigate how host mucus glycan composition interacts with dietary carbohydrate content to influence the composition and expressed functions of a human gut community. The humanized gnotobiotic mice mimic humans with a nonsecretor phenotype due to knockout of their α1-2 fucosyltransferase (Fut2) gene. The fecal microbiota of Fut2(-) mice that lack fucosylated host glycans show decreased alpha diversity relative to Fut2(+) mice and exhibit significant differences in community composition. A glucose-rich plant polysaccharide-deficient (PD) diet exerted a strong effect on the microbiota membership but eliminated the effect of Fut2 genotype. Additionally fecal metabolites predicted host genotype in mice on a polysaccharide-rich standard diet but not on a PD diet. A more detailed mechanistic analysis of these interactions involved colonization of gnotobiotic Fut2(+) and Fut2(-) mice with Bacteroides thetaiotaomicron, a prominent member of the human gut microbiota known to adaptively forage host mucosal glycans when dietary polysaccharides are absent. Within Fut2(-) mice, the B. thetaiotaomicron fucose catabolic pathway was markedly down-regulated, whereas BT4241-4247, an operon responsive to terminal β-galactose, the precursor that accumulates in the Fut2(-) mice, was significantly up-regulated. These changes in B. thetaiotaomicron gene expression were only evident in mice fed a PD diet, wherein B. thetaiotaomicron relies on host mucus consumption. Furthermore, up-regulation of the BT4241-4247 operon was also seen in humanized Fut2(-) mice. Together, these data demonstrate that differences in host genotype that affect the carbohydrate landscape of the distal gut interact with diet to alter the composition and function of resident microbes in a diet-dependent manner.
The human intestine, colonized by a dense community of resident microbes, is a frequent target of bacterial pathogens. Undisturbed, this intestinal microbiota provides protection from bacterial infections. Conversely, disruption of the microbiota with oral antibiotics often precedes the emergence of several enteric pathogens. How pathogens capitalize upon the failure of microbiota-afforded protection is largely unknown. Here we show that two antibiotic-associated pathogens, Salmonella enterica serovar Typhimurium (S. typhimurium) and Clostridium difficile, use a common strategy of catabolizing microbiota-liberated mucosal carbohydrates during their expansion within the gut. S. typhimurium accesses fucose and sialic acid within the lumen of the gut in a microbiota-dependent manner, and genetic ablation of the respective catabolic pathways reduces its competitiveness in vivo. Similarly, C. difficile expansion is aided by microbiota-induced elevation of sialic acid levels in vivo. Colonization of gnotobiotic mice with a sialidase-deficient mutant of Bacteroides thetaiotaomicron, a model gut symbiont, reduces free sialic acid levels resulting in C. difficile downregulating its sialic acid catabolic pathway and exhibiting impaired expansion. These effects are reversed by exogenous dietary administration of free sialic acid. Furthermore, antibiotic treatment of conventional mice induces a spike in free sialic acid and mutants of both Salmonella and C. difficile that are unable to catabolize sialic acid exhibit impaired expansion. These data show that antibiotic-induced disruption of the resident microbiota and subsequent alteration in mucosal carbohydrate availability are exploited by these two distantly related enteric pathogens in a similar manner. This insight suggests new therapeutic approaches for preventing diseases caused by antibiotic-associated pathogens.
The diverse community of microbes that inhabits the human bowel is vitally important to human health. Host-expressed proteins are essential for maintaining this mutualistic relationship and serve as reporters on the status of host-microbiota interaction. Therefore, unbiased and sensitive methods focused on host proteome characterization are needed. Herein we describe a novel method for applying shotgun proteomics to the analysis of feces, focusing on the secreted host proteome. We have conducted the most complete analysis of the extracellular mouse gut proteome to date by employing a gnotobiotic mouse model. Using mice colonized with defined microbial communities of increasing complexity or a complete human microbiota ('humanized'), we show that the complexity of the host stool proteome mirrors the complexity of microbiota composition. We further show that host responses exhibit signatures specific to the different colonization states. We demonstrate feasibility of this approach in human stool samples and provide evidence for a "core" stool proteome as well as personalized host response features. Our method provides a new avenue for noninvasive monitoring of host-microbiota interaction dynamics via host-produced proteins in stool.
While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCer(Bf)), which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (iNKT) cells. We demonstrate that α-GalCer(Bf) has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis.
The human intestine houses a dense microbial ecosystem in which the struggle for nutrients creates a continual and dynamic selective force. Host-produced mucus glycans provide a ubiquitous source of carbon and energy for microbial species. Not surprisingly, many gut resident bacteria have become highly adapted to efficiently consume numerous distinct structures present in host glycans. We propose that sophistication in mucus consumption is a trait most likely to be found in gut residents that have co-evolved with hosts, microbes that have adapted to the complexity associated with the host glycan landscape.
Diet has major effects on the intestinal microbiota, but the exact mechanisms that alter complex microbial communities have been difficult to elucidate. In addition to the direct influence that diet exerts on microbes, changes in microbiota composition and function can alter host functions such as gastrointestinal (GI) transit time, which in turn can further affect the microbiota. We investigated the relationships among diet, GI motility, and the intestinal microbiota using mice that are germ-free (GF) or humanized (ex-GF mice colonized with human fecal microbiota). Analysis of gut motility revealed that humanized mice fed a standard polysaccharide-rich diet had faster GI transit and increased colonic contractility compared with GF mice. Humanized mice with faster transit due to administration of polyethylene glycol or a nonfermentable cellulose-based diet had similar changes in gut microbiota composition, indicating that diet can modify GI transit, which then affects the composition of the microbial community. However, altered transit in mice fed a diet of fermentable fructooligosaccharide indicates that diet can change gut microbial function, which can affect GI transit. Based on studies in humanized mice, diet can affect GI transit through microbiota-dependent or microbiota-independent pathways, depending on the type of dietary change. The effect of the microbiota on transit largely depends on the amount and type (fermentable vs nonfermentable) of polysaccharides present in the diet. These results have implications for disorders that affect GI transit and gut microbial communities, including irritable bowel syndrome and inflammatory bowel disease.
The communities constituting our microbiotas are emerging as mediators of the health-disease continuum. However, deciphering the functional impact of microbial communities on host pathophysiology represents a formidable challenge, due to the heterogeneous distribution of chemical and microbial species within the gastrointestinal (GI) tract. Herein, we apply imaging mass spectrometry (IMS) to localize metabolites from the interaction between the host and colonizing microbiota. This approach complements other molecular imaging methodologies in that analytes need not be known a priori, offering the possibility of untargeted analysis. Localized molecules within the GI tract were then identified in situ by surface sampling with nanodesorption electrospray ionization Fourier transform ion cyclotron resonance-mass spectrometry (nanoDESI FTICR-MS). Products from diverse structural classes were identified including cholesterol-derived lipids, glycans, and polar metabolites. Specific chemical transformations performed by the microbiota were validated with bacteria in culture. This study illustrates how untargeted spatial characterization of metabolites can be applied to the molecular dissection of complex biology in situ.
Bacteroides is a dominant genus within the intestinal microbiota of healthy humans. Key adaptations of the Bacteroides to the dynamic intestinal ecosystem include a diverse repertoire of genes involved in sensing and processing numerous diet- and host-derived polysaccharides. One such adaptation is the carbohydrate-sensing hybrid two-component system (HTCS) family of signalling sensors, which has been widely expanded within the Bacteroides. Using Bacteroides thetaiotaomicron as a model, we have created a chimeric HTCS consisting of the well-characterized sensing domain of one HTCS, BT1754, and the regulatory domain of another HTCS, BT0366, to explore the regulatory capabilities of these molecules. We found that the BT0366 regulatory region directly binds to and mediates induction of the adjacent polysaccharide utilization locus (PUL) using whole-genome transcriptional profiling after inducing signalling through our chimeric protein. We also found that BT0366 activation simultaneously leads to repression of distal PULs involved in mucus carbohydrate consumption. These results suggest a novel mechanism by which an HTCS enforces a nutrient hierarchy within the Bacteroides via induction and repression of multiple PULs. Thus, hybrid two-component systems provide a mechanism for prioritizing consumption of carbohydrates through simultaneous binding and regulation of multiple polysaccharide utilization loci.
Newborns are colonized with an intestinal microbiota shortly after birth, but the factors governing the retention and abundance of specific microbial lineages are unknown. Nursing infants consume human milk oligosaccharides (HMOs) that pass undigested to the distal gut, where they may be digested by microbes. We determined that the prominent neonate gut residents, Bacteroides thetaiotaomicron and Bacteroides fragilis, induce the same genes during HMO consumption that are used to harvest host mucus glycans, which are structurally similar to HMOs. Lacto-N-neotetraose, a specific HMO component, selects for HMO-adapted species such as Bifidobacterium infantis, which cannot use mucus, and provides a selective advantage to B. infantis in vivo when biassociated with B. thetaiotaomicron in the gnotobiotic mouse gut. This indicates that the complex oligosaccharide mixture within HMOs attracts both mutualistic mucus-adapted species and HMO-adapted bifidobacteria to the infant intestine that likely facilitate both milk and future solid food digestion.
In bacterial communities, "tight economic times" are the norm. Of the many challenges bacteria face in making a living, perhaps none are more important than generating energy, maintaining redox balance, and acquiring carbon and nitrogen to synthesize primary metabolites. The ability of bacteria to meet these challenges depends heavily on the rest of their community. Indeed, the most fundamental way in which bacteria communicate is by importing the substrates for metabolism and exporting metabolic end products. As an illustration of this principle, we will travel down a carbohydrate catabolic pathway common to many species of Bacteroides, highlighting the interspecies interactions established (often inevitably) at its key steps. We also discuss the metabolic considerations in maintaining the stability of host-associated microbial communities.
Many infections start with microbial invasion of mucosal surfaces, which are typically colonized by a community of resident microbes. A growing body of literature demonstrates that the resident microbiota plays a significant role in host susceptibility to pathogens. Recent work has largely focused on the considerable effect that the intestinal microbiota can have upon bacterial pathogenesis. These studies reveal many significant gaps in our knowledge about the mechanisms by which the resident community impacts pathogen invasion and the nature of the ensuing host immune response. It is likely that as viral pathogens become the focus of studies that examine microbiota-host interaction, substantial effects of resident communities exerted via diverse mechanisms will be elucidated. Here we provide a perspective of the exciting emerging field that examines how the intestinal microbiota influences host susceptibility to viruses.
It is becoming increasingly clear that diet is one of the major factors that drives the function and composition of the intestinal microbiota. The diet of humans is highly diverse when considering different populations or even a single individual over a relatively short period of time. However, we are just beginning to understand the mechanisms that connect dietary change to intestinal microbiota dynamics. The community of microbes within our distal digestive tract influences numerous aspects of our biology, and aberrant shifts in its composition appear to be associated with several diseases. It is, therefore, necessary to understand how our behaviour and environmental factors, such as changes in diet, impact our intestinal residents. Here we look to recent work to highlight some of the major questions on the horizon for understanding the key role that the Bacteroidetes play in the commerce of dietary polysaccharides within the intestine.
We are never alone. Humans coexist with diverse microbial species that live within and upon us--our so-called microbiota. It is now clear that this microbial community is essentially another organ that plays a fundamental role in human physiology and disease. Basic and translational research efforts have begun to focus on deciphering mechanisms of microbiome function--and learning how to manipulate it to benefit human health. In this Perspective, we discuss therapeutic opportunities in the human microbiome.
The intestinal microbiota impacts many facets of human health and is associated with human diseases. Diet impacts microbiota composition, yet mechanisms that link dietary changes to microbiota alterations remain ill-defined. Here we elucidate the basis of Bacteroides proliferation in response to fructans, a class of fructose-based dietary polysaccharides. Structural and genetic analysis disclosed a fructose-binding, hybrid two-component signaling sensor that controls the fructan utilization locus in Bacteroides thetaiotaomicron. Gene content of this locus differs among Bacteroides species and dictates the specificity and breadth of utilizable fructans. BT1760, an extracellular beta2-6 endo-fructanase, distinguishes B. thetaiotaomicron genetically and functionally, and enables the use of the beta2-6-linked fructan levan. The genetic and functional differences between Bacteroides species are predictive of in vivo competitiveness in the presence of dietary fructans. Gene sequences that distinguish species' metabolic capacity serve as potential biomarkers in microbiomic datasets to enable rational manipulation of the microbiota via diet.
Germ-free mice were maintained on polysaccharide-rich or simple-sugar diets and colonized for 10 days with an organism also found in human guts, Bacteroides thetaiotaomicron, followed by whole-genome transcriptional profiling of bacteria and mass spectrometry of cecal glycans. We found that these bacteria assembled on food particles and mucus, selectively induced outer-membrane polysaccharide-binding proteins and glycoside hydrolases, prioritized the consumption of liberated hexose sugars, and revealed a capacity to turn to host mucus glycans when polysaccharides were absent from the diet. This flexible foraging behavior should contribute to ecosystem stability and functional diversity.