Honors & Awards
NIH Directors New Innovator Award, NIH (2009)
Burroughs Wellcome Fund Investigators in Pathogenesis of Infectious Disease Award, Burroughs Wellcome Fund (2011)
BS, UC Davis, Biochemistry (1996)
PhD, UC San Diego, Biomedical Sciences (2003)
Current Research and Scholarly Interests
We are interested in the basic principles that govern interactions within the intestinal microbiota and between the microbiota and the host. To pursue these aims, we colonize germ-free (gnotobiotic) mice with simplified, model microbial communities, apply systems approaches (e.g. functional genomics), and use genetic tools for the host and microbes to gain mechanistic insight into emergent properties of the host-microbial super-organism.
- Gut Microbiota in Health and Disease
MI 221 (Spr)
- Independent Studies (5)
Prior Year Courses
- Computational Modeling of Microbial Communities
BIOE 115, MI 245 (Spr)
- Computational Modeling of Microbial Communities
Graduate and Fellowship Programs
A small-molecule antivirulence agent for treating Clostridium difficile infection.
Science translational medicine
2015; 7 (306): 306ra148-?
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
Monitoring host responses to the gut microbiota.
2015; 9 (9): 1908-1915
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.The ISME Journal advance online publication, 9 June 2015; doi:10.1038/ismej.2015.93.
View details for DOI 10.1038/ismej.2015.93
View details for PubMedID 26057846
- Monitoring host responses to the gut microbiota ISME JOURNAL 2015; 9 (9): 1908-1915
Gut microbes promote colonic serotonin production through an effect of short-chain fatty acids on enterochromaffin cells
2015; 29 (4): 1395-1403
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.-Reigstad, C. S., Salmonson, C. E., Rainey, III, J. F., Szurszewski, J. H., Linden, D. R., Sonnenburg, J. L., Farrugia, G., Kashyap, P. C. Gut microbes promote colonic serotonin production through an effect of short-chain fatty acids on enterochromaffin cells.
View details for DOI 10.1096/fj.14-259598
View details for Web of Science ID 000354115000025
View details for PubMedID 25550456
- Microbiome engineering. Nature 2015; 518 (7540): S10-?
Metabolome progression during early gut microbial colonization of gnotobiotic mice.
2015; 5: 11589-?
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
Quantitative Imaging of Gut Microbiota Spatial Organization.
Cell host & microbe
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.
View details for DOI 10.1016/j.chom.2015.09.002
View details for PubMedID 26439864
Gut Microbiota-Produced Succinate Promotes C-difficile Infection after Antibiotic Treatment or Motility Disturbance
CELL HOST & MICROBE
2014; 16 (6): 770-777
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.
View details for DOI 10.1016/j.chom.2014.11.003
View details for Web of Science ID 000346211100010
Starving our microbial self: the deleterious consequences of a diet deficient in microbiota-accessible carbohydrates.
2014; 20 (5): 779-786
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.
View details for DOI 10.1016/j.cmet.2014.07.003
View details for PubMedID 25156449
- Starving our Microbial Self: The Deleterious Consequences of a Diet Deficient in Microbiota-Accessible Carbohydrates CELL METABOLISM 2014; 20 (5): 779-786
Reprograming of gut microbiome energy metabolism by the FUT2 Crohn's disease risk polymorphism.
2014; 8 (11): 2193-2206
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.
View details for DOI 10.1038/ismej.2014.64
View details for PubMedID 24781901
- Reprograming of gut microbiome energy metabolism by the FUT2 Crohn's disease risk polymorphism ISME JOURNAL 2014; 8 (11): 2193-2206
The Enteric Two-Step: nutritional strategies of bacterial pathogens within the gut
2014; 16 (7): 993-1003
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.
View details for DOI 10.1111/cmi.12300
View details for Web of Science ID 000337667600002
Gut microbes take their vitamins.
Cell host & microbe
2014; 15 (1): 5-6
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.
View details for DOI 10.1016/j.chom.2013.12.011
View details for PubMedID 24439893
Host-centric Proteomics of Stool: A Novel Strategy Focused on intestinal Responses to the Gut Microbiota
MOLECULAR & CELLULAR PROTEOMICS
2013; 12 (11): 3310-3318
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.
View details for DOI 10.1074/mcp.M113.029967
View details for Web of Science ID 000328816000022
View details for PubMedID 23982161
Genetically dictated change in host mucus carbohydrate landscape exerts a diet-dependent effect on the gut microbiota
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (42): 17059-17064
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
Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens.
2013; 502 (7469): 96-99
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
- Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens NATURE 2013; 502 (7469): 96-?
A metabolomic view of how the human gut microbiota impacts the host metabolome using humanized and gnotobiotic mice
2013; 7 (10): 1933-1943
Defining the functional status of host-associated microbial ecosystems has proven challenging owing to the vast number of predicted genes within the microbiome and relatively poor understanding of community dynamics and community-host interaction. Metabolomic approaches, in which a large number of small molecule metabolites can be defined in a biological sample, offer a promising avenue to 'fingerprint' microbiota functional status. Here, we examined the effects of the human gut microbiota on the fecal and urinary metabolome of a humanized (HUM) mouse using an optimized ultra performance liquid chromatography-mass spectrometry-based method. Differences between HUM and conventional mouse urine and fecal metabolomic profiles support host-specific aspects of the microbiota's metabolomic contribution, consistent with distinct microbial compositions. Comparison of microbiota composition and metabolome of mice humanized with different human donors revealed that the vast majority of metabolomic features observed in donor samples are produced in the corresponding HUM mice, and individual-specific features suggest 'personalized' aspects of functionality can be reconstituted in mice. Feeding the mice a defined, custom diet resulted in modification of the metabolite signatures, illustrating that host diet provides an avenue for altering gut microbiota functionality, which in turn can be monitored via metabolomics. Using a defined model microbiota consisting of one or two species, we show that simplified communities can drive major changes in the host metabolomic profile. Our results demonstrate that metabolomics constitutes a powerful avenue for functional characterization of the intestinal microbiota and its interaction with the host.The ISME Journal advance online publication, 6 June 2013; doi:10.1038/ismej.2013.89.
View details for DOI 10.1038/ismej.2013.89
View details for Web of Science ID 000324869400006
A refined palate: bacterial consumption of host glycans in the gut.
2013; 23 (9): 1038-1046
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.
View details for DOI 10.1093/glycob/cwt040
View details for PubMedID 23720460
- A refined palate: Bacterial consumption of host glycans in the gut GLYCOBIOLOGY 2013; 23 (9): 1038-?
- Production of alpha-Galactosylceramide by a Prominent Member of the Human Gut Microbiota PLOS BIOLOGY 2013; 11 (7)
Complex Interactions Among Diet, Gastrointestinal Transit, and Gut Microbiota in Humanized Mice
2013; 144 (5): 967-977
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
Molecular Analysis of Model Gut Microbiotas by Imaging Mass Spectrometry and Nanodesorption Electrospray Ionization Reveals Dietary Metabolite Transformations
2012; 84 (21): 9259-9267
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
Prioritization of a plant polysaccharide over a mucus carbohydrate is enforced by a Bacteroides hybrid two-component system
2012; 85 (3): 478-491
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.
View details for DOI 10.1111/j.1365-2958.2012.08123.x
View details for Web of Science ID 000306685300007
View details for PubMedID 22686399
Bacteroides in the Infant Gut Consume Milk Oligosaccharides via Mucus-Utilization Pathways
CELL HOST & MICROBE
2011; 10 (5): 507-514
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.
View details for DOI 10.1016/j.chom.2011.10.007
View details for Web of Science ID 000297495100010
View details for PubMedID 22036470
Eating For Two: How Metabolism Establishes lnterspecies Interactions in the Gut
CELL HOST & MICROBE
2011; 10 (4): 336-347
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.
View details for DOI 10.1016/j.chom.2011.10.002
View details for Web of Science ID 000296600700009
View details for PubMedID 22018234
Community Health Care: Therapeutic Opportunities in the Human Microbiome
SCIENCE TRANSLATIONAL MEDICINE
2011; 3 (78)
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.
View details for DOI 10.1126/scitranslmed.3001626
View details for Web of Science ID 000292976400002
View details for PubMedID 21490274
PhyloChip microarray analysis reveals altered gastrointestinal microbial communities in a rat model of colonic hypersensitivity
NEUROGASTROENTEROLOGY AND MOTILITY
2011; 23 (2)
Irritable bowel syndrome (IBS) is a chronic, episodic gastrointestinal disorder that is prevalent in a significant fraction of western human populations; and changes in the microbiota of the large bowel have been implicated in the pathology of the disease.Using a novel comprehensive, high-density DNA microarray (PhyloChip) we performed a phylogenetic analysis of the microbial community of the large bowel in a rat model in which intracolonic acetic acid in neonates was used to induce long lasting colonic hypersensitivity and decreased stool water content and frequency, representing the equivalent of human constipation-predominant IBS.Our results revealed a significantly increased compositional difference in the microbial communities in rats with neonatal irritation as compared with controls. Even more striking was the dramatic change in the ratio of Firmicutes relative to Bacteroidetes, where neonatally irritated rats were enriched more with Bacteroidetes and also contained a different composition of species within this phylum. Our study also revealed differences at the level of bacterial families and species.The PhyloChip is a useful and convenient method to study enteric microflora. Further, this rat model system may be a useful experimental platform to study the causes and consequences of changes in microbial community composition associated with IBS.
View details for DOI 10.1111/j.1365-2982.2010.01637.x
View details for Web of Science ID 000286211600017
View details for PubMedID 21129126
- The intestinal microbiota and viral susceptibility FRONTIERS IN MICROBIOLOGY 2011; 2
Specificity of Polysaccharide Use in Intestinal Bacteroides Species Determines Diet-Induced Microbiota Alterations
2010; 141 (7): 1241-U256
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
- MICROBIOLOGY Genetic pot luck NATURE 2010; 464 (7290): 837-838
Genomic and metabolic studies of the impact of probiotics on a model gut symbiont and host
2006; 4 (12): 2213-2226
Probiotics are deliberately ingested preparations of live bacterial species that confer health benefits on the host. Many of these species are associated with the fermentation of dairy products. Despite their increasing use, the molecular details of the impact of various probiotic preparations on resident members of the gut microbiota and the host are generally lacking. To address this issue, we colonized germ-free mice with Bacteroides thetaiotaomicron, a prominent component of the adult human gut microbiota, and Bifidobacterium longum, a minor member but a commonly used probiotic. Simultaneous whole genome transcriptional profiling of both bacterial species in their gut habitat and of the intestinal epithelium, combined with mass-spectrometric analysis of habitat-associated carbohydrates, revealed that the presence of B. longum elicits an expansion in the diversity of polysaccharides targeted for degradation by B. thetaiotaomicron (e.g., mannose- and xylose-containing glycans), and induces host genes involved in innate immunity. Although the overall transcriptome expressed by B. thetaiotaomicron when it encounters B. longum in the cecum is dependent upon the genetic background of the mouse (as assessed by a mixed analysis of variance [ANOVA] model of co-colonization experiments performed in NMRI and C57BL/6J animals), B. thetaiotaomicron's expanded capacity to utilize polysaccharides occurs independently of host genotype, and is also observed with a fermented dairy product-associated strain, Lactobacillus casei. This gnotobiotic mouse model provides a controlled case study of how a resident symbiont and a probiotic species adapt their substrate utilization in response to one another, and illustrates both the generality and specificity of the relationship between a host, a component of its microbiota, and intentionally consumed microbial species.
View details for DOI 10.1371/journal.pbio.0040413
View details for Web of Science ID 000242789100005
View details for PubMedID 17132046
A hybrid two-component system protein of a prominent human gut symbiont couples glycan sensing in vivo to carbohydrate metabolism
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2006; 103 (23): 8834-8839
Bacteroides thetaiotaomicron is a prominent member of our normal adult intestinal microbial community and a useful model for studying the foundations of human-bacterial mutualism in our densely populated distal gut microbiota. A central question is how members of this microbiota sense nutrients and implement an appropriate metabolic response. B. thetaiotaomicron contains a large number of glycoside hydrolases not represented in our own proteome, plus a markedly expanded collection of hybrid two-component system (HTCS) proteins that incorporate all domains found in classical two-component environmental sensors into one polypeptide. To understand the role of HTCS in nutrient sensing, we used B. thetaiotaomicron GeneChips to characterize their expression in gnotobiotic mice consuming polysaccharide-rich or -deficient diets. One HTCS, BT3172, was selected for further analysis because it is induced in vivo by polysaccharides, and its absence reduces B. thetaiotaomicron fitness in polysaccharide-rich diet-fed mice. Functional genomic and biochemical analyses of WT and BT3172-deficient strains in vivo and in vitro disclosed that alpha-mannosides induce BT3172 expression, which in turn induces expression of secreted alpha-mannosidases. Yeast two-hybrid screens revealed that the cytoplasmic portion of BT3172's sensor domain serves as a scaffold for recruiting glucose-6-phosphate isomerase and dehydrogenase. These interactions are a unique feature of BT3172 and specific for the cytoplasmic face of its sensor domain. Loss of BT3172 reduces glycolytic pathway activity in vitro and in vivo. Thus, this HTCS functions as a metabolic reaction center, coupling nutrient sensing to dynamic regulation of monosaccharide metabolism. An expanded repertoire of HTCS proteins with diversified sensor domains may be one reason for B. thetaiotaomicron's success in our intestinal ecosystem.
View details for DOI 10.1073/pnas.0603249103
View details for Web of Science ID 000238278400046
View details for PubMedID 16735464
Operon prediction without a training set
2005; 21 (7): 880-888
Annotation of operons in a bacterial genome is an important step in determining an organism's transcriptional regulatory program. While extensive studies of operon structure have been carried out in a few species such as Escherichia coli, fewer resources exist to inform operon prediction in newly sequenced genomes. In particular, many extant operon finders require a large body of training examples to learn the properties of operons in the target organism. For newly sequenced genomes, such examples are generally not available; moreover, a model of operons trained on one species may not reflect the properties of other, distantly related organisms. We encountered these issues in the course of predicting operons in the genome of Bacteroides thetaiotaomicron (B.theta), a common anaerobe that is a prominent component of the normal adult human intestinal microbial community.We describe an operon predictor designed to work without extensive training data. We rely on a small set of a priori assumptions about the properties of the genome being annotated that permit estimation of the probability that two adjacent genes lie in a common operon. Predictions integrate several sources of information, including intergenic distance, common functional annotation and a novel formulation of conserved gene order. We validate our predictor both on the known operons of E.coli and on the genome of B.theta, using expression data to evaluate our predictions in the latter.
View details for DOI 10.1093/bioinformatics/bti123
View details for Web of Science ID 000227977800007
View details for PubMedID 15539453
Glycan foraging in vivo by an intestine-adapted bacterial symbiont
2005; 307 (5717): 1955-1959
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.
View details for DOI 10.1126/science.1109051
View details for Web of Science ID 000227957300055
View details for PubMedID 15790854
Getting a grip on things: how do communities of bacterial symbionts become established in our intestine?
2004; 5 (6): 569-573
The gut contains our largest collection of resident microorganisms. One obvious question is how microbial communities establish and maintain themselves within a perfused intestine. The answers, which may come in part from observations made by environmental engineers and glycobiologists, have important implications for immunologists who wish to understand how indigenous microbial communities are accommodated. Here we propose that the mucus gel layer overlying the intestinal epithelium is a key contributor to the structural and functional stability of this microbiota and its tolerance by the host.
View details for DOI 10.1038/ni1079
View details for Web of Science ID 000221664000014
View details for PubMedID 15164016
A uniquely human consequence of domain-specific functional adaptation in a sialic acid-binding receptor
2004; 14 (4): 339-346
Most mammalian cell surfaces display two major sialic acids (Sias), N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans lack Neu5Gc due to a mutation in CMP-Neu5Ac hydroxylase, which occurred after evolutionary divergence from great apes. We describe an apparent consequence of human Neu5Gc loss: domain-specific functional adaptation of Siglec-9, a member of the family of sialic acid-binding receptors of innate immune cells designated the CD33-related Siglecs (CD33rSiglecs). Binding studies on recombinant human Siglec-9 show recognition of both Neu5Ac and Neu5Gc. In striking contrast, chimpanzee and gorilla Siglec-9 strongly prefer binding Neu5Gc. Simultaneous probing of multiple endogenous CD33rSiglecs on circulating blood cells of human, chimp, or gorilla suggests that the binding differences observed for Siglec-9 are representative of multiple CD33rSiglecs. We conclude that Neu5Ac-binding ability of at least some human CD33rSiglecs is a derived state selected for following loss of Neu5Gc in the hominid lineage. These data also indicate that endogenous Sias (rather than surface Sias of bacterial pathogens) are the functional ligands of CD33rSiglecs and suggest that the endogenous Sia landscape is the major factor directing evolution of CD33rSiglec binding specificity. Exon-1-encoded Sia-recognizing domains of human and ape Siglec-9 share only approximately 93-95% amino acid identity. In contrast, the immediately adjacent intron and exon 2 have the approximately 98-100% identity typically observed among these species. Together, our findings suggest ongoing adaptive evolution specific to the Sia-binding domain, possibly of an episodic nature. Such domain-specific divergences should also be considered in upcoming comparisons of human and chimpanzee genomes.
View details for DOI 10.1093/glycob/cwh039
View details for Web of Science ID 000220896000006
View details for PubMedID 14693915
Characterization of the acid stability of glycosidically linked neuraminic acid - Use in detecting de-N-acetyl-gangliosides in human melanoma
JOURNAL OF BIOLOGICAL CHEMISTRY
2002; 277 (20): 17502-17510
The glycosidic linkage of sialic acids is much more sensitive to acid hydrolysis than those of other monosaccharides in vertebrates. The commonest sialic acids in nature are neuraminic acid (Neu)-based and are typically N-acylated at the C5 position. Unsubstituted Neu is thought to occur on native gangliosides of certain tumors and cell lines, and synthetic de-N-acetyl-gangliosides have potent biological properties in vitro. However, claims for their natural existence are based upon monoclonal antibodies and pulse-chase experiments, and there have been no reports of their chemical detection. Here we report that one of these antibodies shows nonspecific cross-reactivity with a polypeptide epitope, further emphasizing the need for definitive chemical proof of unsubstituted Neu on naturally occurring gangliosides. While pursuing this, we found that alpha2-3-linked Neu on chemically de-N-acetylated G(M3) ganglioside resists acid hydrolysis under conditions where the N-acetylated form is completely labile. To ascertain the generality of this finding, we investigated the stability of glycosidically linked alpha- and beta-methyl glycosides of Neu. Using NMR spectroscopy to monitor glycosidic linkage hydrolysis, we find that only 47% of Neualpha2Me is hydrolyzed after 3 h in 10 mm HCl at 80 degrees C, whereas Neu5Acalpha2Me is 95% hydrolyzed after 20 min under the same conditions. Notably, Neubeta2Me is hydrolyzed even slower than Neualpha2Me, indicating that acid resistance is a general property of glycosidically linked Neu. Taking advantage of this, we modified classical purification techniques for de-N-acetyl-ganglioside isolation using acid to first eliminate conventional gangliosides. We also introduce a phospholipase-based approach to remove contaminating phospholipids that previously hindered efforts to study de-N-acetyl-gangliosides. The partially purified sample can then be N-propionylated, allowing acid release and mass spectrometric detection of any originally existing Neu as Neu5Pr. These advances allowed us to detect covalently bound Neu in lipid extracts of a human melanoma tumor, providing the first chemical proof for naturally occurring de-N-acetyl-gangliosides.
View details for DOI 10.1074/jbc.M110867200
View details for Web of Science ID 000175685100016
View details for PubMedID 11884388
- Effects of sialic acid substitutions on recognition by Sambucus nigra agglutinin and Maackia amurensis hemagglutinin ANALYTICAL BIOCHEMISTRY 2002; 303 (1): 98-104
De-N-acetyl-gangliosides in humans: Unusual subcellular distribution of a novel tumor antigen
1999; 59 (6): 1337-1346
The disialoganglioside GD3 is a major antigen in human melanomas that can undergo 9-O-acetylation of the outer sialic acid (giving 9-OAc-GD3). Monoclonal antibody SGR37 detects a different modification of the GD3, de-N-acetylation of the 5-N-acetyl group (giving de-N-Ac-GD3). We found that conventional immunohistochemistry of the SGR37 antigen is limited by a reduction in reactivity upon fixation with aldehydes (which presumably react with the free amino group) or with organic reagents (which can extract glycolipids). We optimized conditions for detection of this antigen in unfixed frozen tissue sections and studied its distribution in human tissues and tumors. It is expressed at low levels in a few blood vessels, infiltrating mononuclear cells in the skin and colon, and at moderate levels in skin melanocytes. In contrast, the antigen accumulates at high levels in many melanomas and in some lymphomas but not in carcinomas. In positive melanomas, expression is sometimes more intense and widespread than that of GD3. Both 9-O-acetylation and de-N-acetylation of GD3 seem to occur after its initial biosynthesis. Isotype-matched antibodies against GD3, 9-O-acetyl-GD3 and de-N-acetyl-GD3 were used to compare their subcellular localization and trafficking. 9-O-acetyl-GD3 colocalizes with GD3 predominantly on the cell surface and partly in lysosomal compartments. In contrast, de-N-acetyl-GD3 has a diffuse intracellular location. Adsorptive endocytosis of antibodies indicates that whereas GD3 remains predominantly on the cell surface, de-N-acetyl-GD3 is efficiently internalized into a compartment that is distinct from lysosomes. Rounding up of melanoma cells occurring during growth in culture is associated with relocation of the internal pool of de-N-acetyl-GD3 to the cell surface. Thus, a minor modification of the polar head group of a tumor-associated glycosphingolipid can markedly affect the subcellular localization and trafficking of the whole molecule. The high levels of the SGR37 antigen in melanomas and lymphomas, its selective endocytosis from the cell surface, and its relocation to the cell surface of rounded up cells suggest potential uses in diagnostic or therapeutic approaches to these diseases.
View details for Web of Science ID 000079125200028
View details for PubMedID 10096568
Resolution of subunit interactions and cytoplasmic subcomplexes of the yeast vacuolar proton-translocating ATPase
JOURNAL OF BIOLOGICAL CHEMISTRY
1996; 271 (17): 10397-10404
The vacuolar proton-translocating ATPase is the principal energization mechanism that enables the yeast vacuole to perform most of its physiological functions. We have undertaken an examination of subunit-subunit interactions and assembly states of this enzyme. Yeast two-hybrid data indicate that Vma1p and Vma2p interact with each other and that Vma4p interacts with itself. Three-hybrid data indicate that the Vma4p self-interaction is stabilized by both Vma1p and Vma2p. Native gel electrophoresis reveals numerous partial complexes not previously described. In addition to a large stable cytoplasmic complex seen in wild-type, Deltavma3 and Deltavma5 strains, we see partial complexes in the Deltavma4 and Deltavma7 strains. All larger complexes are lost in the Deltavma1, Deltavma2, and Deltavma8 strains. We designate the large complex seen in wild-type cells containing at least subunits Vma1p, Vma2p, Vma4p, Vma7p, and Vma8p as the definitive V1 complex.
View details for Web of Science ID A1996UG25700085
View details for PubMedID 8626613