Current Role at Stanford
Maintain and operate Gnotobiotic research facility.
Maintain and operate Gnotobiotic research facility.
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 invitro. Monocolonization of mice with mutant BtDelta2157 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.
View details for DOI 10.1016/j.cell.2020.01.023
View details for PubMedID 32084341
The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 μM and are known to block the growth of Clostridium difficile1, promote hepatocellular carcinoma2 and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. 3). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids4; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool.
View details for DOI 10.1038/s41586-020-2396-4
View details for PubMedID 32555455
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.
View details for DOI 10.1126/science.aav1282
View details for PubMedID 31831639
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.
View details for PubMedID 30808756
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.
View details for DOI 10.1016/j.chom.2019.10.011
View details for PubMedID 31726029
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 invitro, 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.
View details for PubMedID 29906449
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.
View details for PubMedID 29794055
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.
View details for PubMedID 29168502
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.
View details for PubMedID 27822551
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.
View details for DOI 10.1038/nature16504
View details for PubMedID 26762459
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.
View details for DOI 10.1126/scitranslmed.aac9103
View details for PubMedID 26400909
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.
View details for DOI 10.1038/srep11589
View details for PubMedID 26118551
View details for PubMedCentralID PMC4484351
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.
View details for DOI 10.1073/pnas.1306070110
View details for Web of Science ID 000325634200076
View details for PubMedID 24062455
View details for PubMedCentralID PMC3800993
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.
View details for DOI 10.1038/nature12503
View details for PubMedID 23995682
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.
View details for DOI 10.1053/j.gastro.2013.01.047
View details for Web of Science ID 000317813900026
View details for PubMedID 23380084
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.
View details for DOI 10.1021/ac302039u
View details for Web of Science ID 000310664600055
View details for PubMedID 23009651
View details for PubMedCentralID PMC3711173
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.
View details for DOI 10.1016/j.cell.2010.05.005
View details for Web of Science ID 000279148100020
View details for PubMedID 20603004
View details for PubMedCentralID PMC2900928
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