RESUMO
Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and ß2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.
Assuntos
Doenças Cardiovasculares/sangue , Microbioma Gastrointestinal/genética , Glutamina/análogos & derivados , Trombose/metabolismo , Animais , Artérias/lesões , Artérias/metabolismo , Artérias/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Plaquetas/metabolismo , Plaquetas/microbiologia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/microbiologia , Doenças Cardiovasculares/patologia , Morte Súbita Cardíaca/patologia , Glutamina/sangue , Glutamina/genética , Humanos , Masculino , Metaboloma/genética , Metabolômica/métodos , Camundongos , Infarto do Miocárdio/sangue , Infarto do Miocárdio/microbiologia , Ativação Plaquetária/genética , Receptores Adrenérgicos alfa/sangue , Receptores Adrenérgicos alfa/genética , Receptores Adrenérgicos beta/sangue , Receptores Adrenérgicos beta/genética , Fatores de Risco , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/microbiologia , Acidente Vascular Cerebral/patologia , Trombose/genética , Trombose/microbiologia , Trombose/patologiaRESUMO
Histone-modifying enzymes regulate transcription and are sensitive to availability of endogenous small-molecule metabolites, allowing chromatin to respond to changes in environment. The gut microbiota produces a myriad of metabolites that affect host physiology and susceptibility to disease; however, the underlying molecular events remain largely unknown. Here we demonstrate that microbial colonization regulates global histone acetylation and methylation in multiple host tissues in a diet-dependent manner: consumption of a "Western-type" diet prevents many of the microbiota-dependent chromatin changes that occur in a polysaccharide-rich diet. Finally, we demonstrate that supplementation of germ-free mice with short-chain fatty acids, major products of gut bacterial fermentation, is sufficient to recapitulate chromatin modification states and transcriptional responses associated with colonization. These findings have profound implications for understanding the complex functional interactions between diet, gut microbiota, and host health.
Assuntos
Dieta Ocidental , Epigênese Genética , Ácidos Graxos Voláteis/metabolismo , Microbioma Gastrointestinal/fisiologia , Tecido Adiposo/enzimologia , Tecido Adiposo/metabolismo , Animais , Colo/enzimologia , Colo/metabolismo , Metilação de DNA , Histonas/genética , Histonas/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de ÓrgãosRESUMO
AIMS: Precision microbiome modulation as a novel treatment strategy is a rapidly evolving and sought goal. The aim of this study is to determine relationships among systemic gut microbial metabolite levels and incident cardiovascular disease risks to identify gut microbial pathways as possible targets for personalized therapeutic interventions. METHODS AND RESULTS: Stable isotope dilution mass spectrometry methods to quantitatively measure aromatic amino acids and their metabolites were used to examine sequential subjects undergoing elective diagnostic cardiac evaluation in two independent cohorts with longitudinal outcome data [US (n = 4000) and EU (n = 833) cohorts]. It was also used in plasma from humans and mice before vs. after a cocktail of poorly absorbed antibiotics to suppress gut microbiota. Multiple aromatic amino acid-derived metabolites that originate, at least in part, from gut bacteria are associated with incident (3-year) major adverse cardiovascular event (MACE) risks (myocardial infarction, stroke, or death) and all-cause mortality independent of traditional risk factors. Key gut microbiota-derived metabolites associated with incident MACE and poorer survival risks include: (i) phenylacetyl glutamine and phenylacetyl glycine (from phenylalanine); (ii) p-cresol (from tyrosine) yielding p-cresol sulfate and p-cresol glucuronide; (iii) 4-OH-phenyllactic acid (from tyrosine) yielding 4-OH-benzoic acid and 4-OH-hippuric acid; (iv) indole (from tryptophan) yielding indole glucuronide and indoxyl sulfate; (v) indole-3-pyruvic acid (from tryptophan) yielding indole-3-lactic acid and indole-3-acetyl-glutamine, and (vi) 5-OH-indole-3-acetic acid (from tryptophan). CONCLUSION: Key gut microbiota-generated metabolites derived from aromatic amino acids independently associated with incident adverse cardiovascular outcomes are identified, and thus will help focus future studies on gut-microbial metabolic outputs relevant to host cardiovascular health.
Assuntos
Microbioma Gastrointestinal , Infarto do Miocárdio , Humanos , Camundongos , Animais , Aminoácidos Aromáticos/metabolismo , Triptofano , Glutamina , Glucuronídeos , Indóis/metabolismo , Progressão da Doença , TirosinaRESUMO
RATIONALE: Gut microbes influence cardiovascular disease and thrombosis risks through the production of trimethylamine N-oxide (TMAO). Microbiota-dependent generation of trimethylamine (TMA)-the precursor to TMAO-is rate limiting in the metaorganismal TMAO pathway in most humans and is catalyzed by several distinct microbial choline TMA-lyases, including the proteins encoded by the cutC/D (choline utilization C/D) genes in multiple human commensals. OBJECTIVE: Direct demonstration that the gut microbial cutC gene is sufficient to transmit enhanced platelet reactivity and thrombosis potential in a host via TMA/TMAO generation has not yet been reported. METHODS AND RESULTS: Herein, we use gnotobiotic mice and a series of microbial colonization studies to show that microbial cutC-dependent TMA/TMAO production is sufficient to transmit heightened platelet reactivity and thrombosis potential in a host. Specifically, we examine in vivo thrombosis potential employing germ-free mice colonized with either high TMA-producing stable human fecal polymcrobial communities or a defined CutC-deficient background microbial community coupled with a CutC-expressing human commensal±genetic disruption of its cutC gene (ie, Clostridium sporogenes Δ cutC). CONCLUSIONS: Collectively, these studies point to the microbial choline TMA-lyase pathway as a rational molecular target for the treatment of atherothrombotic heart disease.
Assuntos
Proteínas de Bactérias/metabolismo , Transplante de Microbiota Fecal , Liases/metabolismo , Ativação Plaquetária , Trombose/microbiologia , Adulto , Animais , Proteínas de Bactérias/genética , Colina/metabolismo , Clostridium/enzimologia , Clostridium/genética , Feminino , Microbioma Gastrointestinal , Humanos , Liases/genética , Masculino , Metilaminas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Trombose/sangueRESUMO
BACKGROUND: An issue associated with efficient bioethanol production is the fact that the desired product is toxic to the biocatalyst. Among other effects, ethanol has previously been found to influence the membrane of E. coli in a dose-dependent manner and induce changes in the lipid composition of the plasma membrane. We describe here the characterization of a collection of ethanol-tolerant strains derived from the ethanologenic Escherichia coli strain FBR5. RESULTS: Membrane permeability assays indicate that many of the strains in the collection have alterations in membrane permeability and/or responsiveness of the membrane to environmental changes such as temperature shifts or ethanol exposure. However, analysis of the strains by gas chromatography and mass spectrometry revealed no qualitative changes in the acyl chain composition of membrane lipids in response to ethanol or temperature. To determine whether these strains contain any mutations that might contribute to ethanol tolerance or changes in membrane permeability, we sequenced the entire genome of each strain. Unexpectedly, none of the strains displayed mutations in genes known to control membrane lipid synthesis, and a few strains carried no mutations at all. Interestingly, we found that four independently-isolated strains acquired an identical C â A (V244 V) silent mutation in the ferric citrate transporter gene fecA. Further, we demonstrated that either a deletion of fecA or over-expression of fecA can confer increased ethanol survival, suggesting that any misregulation of fecA expression affects the cellular response to ethanol. CONCLUSIONS: The fact that no mutations were observed in several ethanol-tolerant strains suggested that epigenetic mechanisms play a role in E. coli ethanol tolerance and membrane permeability. Our data also represent the first direct phenotypic evidence that the fecA gene plays a role in ethanol tolerance. We propose that the recurring silent mutation may exert an effect on phenotype by altering RNA-mediated regulation of fecA expression.
Assuntos
Tolerância a Medicamentos/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/toxicidade , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Loci Gênicos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Mutação Silenciosa , Temperatura , Sequenciamento Completo do GenomaRESUMO
Recent studies show gut microbiota-dependent metabolism of dietary phenylalanine into phenylacetic acid (PAA) is critical in phenylacetylglutamine (PAGln) production, a metabolite linked to atherosclerotic cardiovascular disease (ASCVD). Accordingly, microbial enzymes involved in this transformation are of interest. Using genetic manipulation in selected microbes and monocolonization experiments in gnotobiotic mice, we identify two distinct gut microbial pathways for PAA formation; one is catalyzed by phenylpyruvate:ferredoxin oxidoreductase (PPFOR) and the other by phenylpyruvate decarboxylase (PPDC). PPFOR and PPDC play key roles in gut bacterial PAA production via oxidative and non-oxidative phenylpyruvate decarboxylation, respectively. Metagenomic analyses revealed a significantly higher abundance of both pathways in gut microbiomes of ASCVD patients compared with controls. The present studies show a role for these two divergent microbial catalytic strategies in the meta-organismal production of PAGln. Given the numerous links between PAGln and ASCVD, these findings will assist future efforts to therapeutically target PAGln formation in vivo.
Assuntos
Doenças Cardiovasculares , Microbioma Gastrointestinal , Camundongos , Animais , GlutaminaRESUMO
p-Cresol sulfate (pCS) and indoxyl sulfate (IS), gut microbiome-derived metabolites, are traditionally associated with cardiovascular disease (CVD) risks in the setting of impaired kidney function. While pharmacologic provision of pCS or IS can promote pro-thrombotic phenotypes, neither the microbial enzymes involved nor direct gut microbial production have been linked to CVD. Untargeted metabolomics was performed on a discovery cohort (n = 1,149) with relatively preserved kidney function, followed by stable isotope-dilution mass spectrometry quantification of pCS and IS in an independent validation cohort (n = 3,954). Genetic engineering of human commensals to produce p-cresol and indole gain-of-function and loss-of-function mutants, followed by colonization of germ-free mice, and studies on host thrombosis were performed. Systemic pCS and IS levels were independently associated with all-cause mortality. Both in vitro and within colonized germ-free mice p-cresol productions were recapitulated by collaboration of two organisms: a Bacteroides strain that converts tyrosine to 4-hydroxyphenylacetate, and a Clostridium strain that decarboxylates 4-hydroxyphenylacetate to p-cresol. We then engineered a single organism, Bacteroides thetaiotaomicron, to produce p-cresol, indole, or both metabolites. Colonizing germ-free mice with engineered strains, we show the gut microbial genes for p-cresol (hpdBCA) and indole (tryptophanase) are sufficient to confer a pro-thrombotic phenotype in vivo. Moreover, human fecal metagenomics analyses show that abundances of hpdBCA and tryptophanase are associated with CVD. These studies show that pCS and IS, two abundant microbiome-derived metabolites, play a broader potential role in CVD than was previously known. They also suggest that therapeutic targeting of gut microbial p-cresol- and indole-producing pathways represent rational targets for CVD.IMPORTANCEAlterations in gut microbial composition and function have been linked to numerous diseases. Identifying microbial pathways responsible for producing molecules that adversely impact the host is an important first step in the development of therapeutic interventions. Here, we first use large-scale clinical observations to link blood levels of defined microbial products to cardiovascular disease risks. Notably, the previously identified uremic toxins p-cresol sulfate and indoxyl sulfate were shown to predict 5-year mortality risks. After identifying the microbes and microbial enzymes involved in the generation of these uremic toxins, we used bioengineering technologies coupled with colonization of germ-free mice to show that the gut microbial genes that generate p-cresol and indole are sufficient to confer p-cresol sulfate and indoxyl sulfate formation, and a pro-thrombotic phenotype in vivo. The findings and tools developed serve as a critical step in both the study and targeting of these gut microbial pathways in vivo.
RESUMO
BACKGROUND: The gut microbiota-dependent metabolite phenylacetylgutamine (PAGln) is both associated with atherothrombotic heart disease in humans, and mechanistically linked to cardiovascular disease pathogenesis in animal models via modulation of adrenergic receptor signaling. METHODS: Here we examined both clinical and mechanistic relationships between PAGln and heart failure (HF). First, we examined associations among plasma levels of PAGln and HF, left ventricular ejection fraction, and N-terminal pro-B-type natriuretic peptide in 2 independent clinical cohorts of subjects undergoing coronary angiography in tertiary referral centers (an initial discovery US Cohort, n=3256; and a validation European Cohort, n=829). Then, the impact of PAGln on cardiovascular phenotypes relevant to HF in cultured cardiomyoblasts, and in vivo were also examined. RESULTS: Circulating PAGln levels were dose-dependently associated with HF presence and indices of severity (reduced ventricular ejection fraction, elevated N-terminal pro-B-type natriuretic peptide) independent of traditional risk factors and renal function in both cohorts. Beyond these clinical associations, mechanistic studies showed both PAGln and its murine counterpart, phenylacetylglycine, directly fostered HF-relevant phenotypes, including decreased cardiomyocyte sarcomere contraction, and B-type natriuretic peptide gene expression in both cultured cardiomyoblasts and murine atrial tissue. CONCLUSIONS: The present study reveals the gut microbial metabolite PAGln is clinically and mechanistically linked to HF presence and severity. Modulating the gut microbiome, in general, and PAGln production, in particular, may represent a potential therapeutic target for modulating HF. REGISTRATION: URL: https://clinicaltrials.gov/; Unique identifier: NCT00590200 and URL: https://drks.de/drks_web/; Unique identifier: DRKS00020915.
Assuntos
Microbioma Gastrointestinal , Insuficiência Cardíaca , Disfunção Ventricular Esquerda , Animais , Humanos , Camundongos , Peptídeo Natriurético Encefálico , Volume Sistólico/fisiologia , Função Ventricular EsquerdaRESUMO
The heightened cardiovascular disease (CVD) risk observed among omnivores is thought to be linked, in part, to gut microbiota-dependent generation of trimethylamine-N-oxide (TMAO) from L-carnitine, a nutrient abundant in red meat. Gut microbial transformation of L-carnitine into trimethylamine (TMA), the precursor of TMAO, occurs via the intermediate γ-butyrobetaine (γBB). However, the interrelationship of γBB, red meat ingestion and CVD risks, as well as the gut microbial genes responsible for the transformation of γBB to TMA, are unclear. In the present study, we show that plasma γBB levels in individuals from a clinical cohort (n = 2,918) are strongly associated with incident CVD event risks. Culture of human faecal samples and microbial transplantation studies in gnotobiotic mice with defined synthetic communities showed that the introduction of Emergencia timonensis, a human gut microbe that can metabolize γBB into TMA, is sufficient to complete the carnitine â γBB â TMA transformation, elevate TMAO levels and enhance thrombosis potential in recipients after arterial injury. RNA-sequencing analyses of E. timonensis identified a six-gene cluster, herein named the γBB utilization (gbu) gene cluster, which is upregulated in response to γBB. Combinatorial cloning and functional studies identified four genes (gbuA, gbuB, gbuC and gbuE) that are necessary and sufficient to recapitulate the conversion of γBB to TMA when coexpressed in Escherichia coli. Finally, reanalysis of samples (n = 113) from a clinical, randomized diet, intervention study showed that the abundance of faecal gbuA correlates with plasma TMAO and a red meat-rich diet. Our findings reveal a microbial gene cluster that is critical to dietary carnitine â γBB â TMA â TMAO transformation in hosts and contributes to CVD risk.
Assuntos
Doenças Cardiovasculares/genética , Carnitina/sangue , Carnitina/metabolismo , Microbioma Gastrointestinal/fisiologia , Genes Bacterianos/genética , Família Multigênica , Carne Vermelha , Animais , Doenças Cardiovasculares/sangue , Clostridiales/genética , Clostridiales/metabolismo , Fezes/microbiologia , Feminino , Vida Livre de Germes , Humanos , Metilaminas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estudos Observacionais como AssuntoRESUMO
Clinical studies have demonstrated associations between circulating levels of the gut-microbiota-derived metabolite trimethylamine-N-oxide (TMAO) and stroke incident risk. However, a causal role of gut microbes in stroke has not yet been demonstrated. Herein we show that gut microbes, through dietary choline and TMAO generation, directly impact cerebral infarct size and adverse outcomes following stroke. Fecal microbial transplantation from low- versus high-TMAO-producing human subjects into germ-free mice shows that both TMAO generation and stroke severity are transmissible traits. Furthermore, employing multiple murine stroke models and transplantation of defined microbial communities with genetically engineered human commensals into germ-free mice, we demonstrate that the microbial cutC gene (an enzymatic source of choline-to-TMA transformation) is sufficient to transmit TMA/TMAO production, heighten cerebral infarct size, and lead to functional impairment. We thus reveal that gut microbiota in general, specifically the metaorganismal TMAO pathway, directly contributes to stroke severity.
Assuntos
Bactérias/metabolismo , Microbioma Gastrointestinal , Metilaminas/metabolismo , Óxidos/metabolismo , Acidente Vascular Cerebral/microbiologia , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Colina/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Índice de Gravidade de Doença , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologiaRESUMO
The gut-microbe-derived metabolite trimethylamine N-oxide (TMAO) is increased by insulin resistance and associated with several sequelae of metabolic syndrome in humans, including cardiovascular, renal, and neurodegenerative disease. The mechanism by which TMAO promotes disease is unclear. We now reveal the endoplasmic reticulum stress kinase PERK (EIF2AK3) as a receptor for TMAO: TMAO binds to PERK at physiologically relevant concentrations; selectively activates the PERK branch of the unfolded protein response; and induces the transcription factor FoxO1, a key driver of metabolic disease, in a PERK-dependent manner. Furthermore, interventions to reduce TMAO, either by manipulation of the gut microbiota or by inhibition of the TMAO synthesizing enzyme, flavin-containing monooxygenase 3, can reduce PERK activation and FoxO1 levels in the liver. Taken together, these data suggest TMAO and PERK may be central to the pathogenesis of the metabolic syndrome.
Assuntos
Síndrome Metabólica/metabolismo , Metilaminas/metabolismo , eIF-2 Quinase/metabolismo , Animais , Microbioma Gastrointestinal/fisiologia , Células HEK293 , Células Hep G2 , Humanos , Indóis/farmacologia , Resistência à Insulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Oxigenases/antagonistas & inibidoresRESUMO
Mounting evidence suggests that environmental stress experienced in utero (for example, maternal nutritional deficits) establishes a predisposition in the newborn to the development of chronic diseases later in life. This concept is often referred to as the "fetal origins hypothesis" or "developmental origins of health and disease". Since its first proposal, epigenetics has emerged as an underlying mechanism explaining how environmental cues become gestationally "encoded". Many of the enzymes that impart and maintain epigenetic modifications are highly sensitive to nutrient availability, which can be influenced by the metabolic activities of the intestinal microbiota. Therefore, the maternal microbiome has the potential to influence epigenetics in utero and modulate offspring's long-term health trajectories. Here we summarize the current understanding of the interactions that occur between the maternal gut microbiome and the essential nutrient choline, that is not only required for fetal development and epigenetic regulation but is also a growth substrate for some microbes. Bacteria able to metabolize choline benefit from the presence of this nutrient and compete with the host for its access, which under extreme conditions may elicit signatures of choline deficiency. Another consequence of bacterial choline metabolism is the accumulation of the pro-inflammatory, pro-thrombotic metabolite trimethylamine-N-oxide (TMAO). Finally, we discuss how these different facets of microbial choline metabolism may influence infant development and health trajectories via epigenetic mechanisms and more broadly place a call to action to better understand how maternal microbial metabolism can shape their offspring's propensity to chronic disease development later in life.
Assuntos
Colina/metabolismo , Epigênese Genética , Microbioma Gastrointestinal/fisiologia , Exposição Materna , Metilaminas/metabolismo , Nutrientes/metabolismo , Oxidantes/metabolismo , Bactérias/metabolismo , Feminino , HumanosRESUMO
BACKGROUND: Trimethylamine N-oxide (TMAO), a small molecule produced by the metaorganismal metabolism of dietary choline, has been implicated in human disease pathogenesis, including known risk factors for Alzheimer's disease (AD), such as metabolic, cardiovascular, and cerebrovascular disease. METHODS: In this study, we tested whether TMAO is linked to AD by examining TMAO levels in cerebrospinal fluid (CSF) collected from a large sample (n = 410) of individuals with Alzheimer's clinical syndrome (n = 40), individuals with mild cognitive impairment (MCI) (n = 35), and cognitively-unimpaired individuals (n = 335). Linear regression analyses were used to determine differences in CSF TMAO between groups (controlling for age, sex, and APOE ε4 genotype), as well as to determine relationships between CSF TMAO and CSF biomarkers of AD (phosphorylated tau and beta-amyloid) and neuronal degeneration (total tau, neurogranin, and neurofilament light chain protein). RESULTS: CSF TMAO is higher in individuals with MCI and AD dementia compared to cognitively-unimpaired individuals, and elevated CSF TMAO is associated with biomarkers of AD pathology (phosphorylated tau and phosphorylated tau/Aß42) and neuronal degeneration (total tau and neurofilament light chain protein). CONCLUSIONS: These findings provide additional insight into gut microbial involvement in AD and add to the growing understanding of the gut-brain axis.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/microbiologia , Microbioma Gastrointestinal , Metilaminas/líquido cefalorraquidiano , Idoso , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Disfunção Cognitiva/líquido cefalorraquidiano , Disfunção Cognitiva/microbiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidianoRESUMO
BACKGROUND: Convenient, reproducible, and rapid preservation of unique biological specimens is pivotal to their use in microbiome analyses. As an increasing number of human studies incorporate the gut microbiome in their design, there is a high demand for streamlined sample collection and storage methods that are amenable to different settings and experimental needs. While several commercial kits address collection/shipping needs for sequence-based studies, these methods do not preserve samples properly for studies that require viable microbes. RESULTS: We describe the Fecal Aliquot Straw Technique (FAST) of fecal sample processing for storage and subsampling. This method uses a straw to collect fecal material from samples recently voided or preserved at low temperature but not frozen (i.e., 4 °C). Different straw aliquots collected from the same sample yielded highly reproducible communities as disclosed by 16S rRNA gene sequencing; operational taxonomic units that were lost, or gained, between the two aliquots represented very low-abundance taxa (i.e., < 0.3% of the community). FAST-processed samples inoculated into germ-free animals resulted in gut communities that retained on average ~ 80% of the donor's bacterial community. Assessment of choline metabolism and trimethylamine-N-oxide accumulation in transplanted mice suggests large interpersonal variation. CONCLUSIONS: Overall, FAST allows for repetitive subsampling without thawing of the specimens and requires minimal supplies and storage space, making it convenient to utilize both in the lab and in the field. FAST has the potential to advance microbiome research through easy, reproducible sample processing.
Assuntos
Bactérias/classificação , Bactérias/genética , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Metilaminas/metabolismo , Manejo de Espécimes/métodos , Animais , Bactérias/isolamento & purificação , Sequência de Bases , Humanos , Camundongos , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Humans with metabolic and inflammatory diseases frequently harbour lower levels of butyrate-producing bacteria in their gut. However, it is not known whether variation in the levels of these organisms is causally linked with disease development and whether diet modifies the impact of these bacteria on health. Here we show that a prominent gut-associated butyrate-producing bacterial genus (Roseburia) is inversely correlated with atherosclerotic lesion development in a genetically diverse mouse population. We use germ-free apolipoprotein E-deficient mice colonized with synthetic microbial communities that differ in their capacity to generate butyrate to demonstrate that Roseburia intestinalis interacts with dietary plant polysaccharides to: impact gene expression in the intestine, directing metabolism away from glycolysis and toward fatty acid utilization; lower systemic inflammation; and ameliorate atherosclerosis. Furthermore, intestinal administration of butyrate reduces endotoxaemia and atherosclerosis development. Together, our results illustrate how modifiable diet-by-microbiota interactions impact cardiovascular disease, and suggest that interventions aimed at increasing the representation of butyrate-producing bacteria may provide protection against atherosclerosis.
Assuntos
Aterosclerose , Clostridiales/metabolismo , Dieta , Microbioma Gastrointestinal , Intestinos/microbiologia , Animais , Apolipoproteínas E/genética , Aterosclerose/tratamento farmacológico , Aterosclerose/patologia , Butiratos/metabolismo , Butiratos/farmacologia , Doenças Cardiovasculares , Clostridiales/genética , Colo/metabolismo , Colo/microbiologia , Carboidratos da Dieta/metabolismo , Modelos Animais de Doenças , Endotoxemia , Metabolismo Energético , Ácidos Graxos/metabolismo , Fezes/microbiologia , Expressão Gênica , Vida Livre de Germes , Masculino , Metaboloma , Camundongos , Camundongos Knockout , RNA Ribossômico 16S/genéticaRESUMO
Triclosan (TCS) is a high-volume chemical used as an antimicrobial ingredient in more than 2000 consumer products, such as toothpaste, cosmetics, kitchenware, and toys. We report that brief exposure to TCS, at relatively low doses, causes low-grade colonic inflammation, increases colitis, and exacerbates colitis-associated colon cancer in mice. Exposure to TCS alters gut microbiota in mice, and its proinflammatory effect is attenuated in germ-free mice. In addition, TCS treatment increases activation of Toll-like receptor 4 (TLR4) signaling in vivo and fails to promote colitis in Tlr4-/- mice. Together, our results demonstrate that this widely used antimicrobial ingredient could have adverse effects on colonic inflammation and associated colon tumorigenesis through modulation of the gut microbiota and TLR4 signaling. Together, these results highlight the need to reassess the effects of TCS on human health and potentially update policies regulating the use of this widely used antimicrobial.
Assuntos
Anti-Infecciosos/efeitos adversos , Carcinogênese/patologia , Colite/complicações , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Inflamação/induzido quimicamente , Animais , Colite/microbiologia , Colite/patologia , Colo/microbiologia , Neoplasias do Colo/microbiologia , Neoplasias do Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Microbioma Gastrointestinal/efeitos dos fármacos , Inflamação/microbiologia , Inflamação/patologia , Masculino , Metaboloma , Camundongos Endogâmicos C57BL , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Triclosan/efeitos adversosRESUMO
Using an untargeted metabolomics approach in initial (N = 99 subjects) and replication cohorts (N = 1,162), we discovered and structurally identified a plasma metabolite associated with cardiovascular disease (CVD) risks, N6,N6,N6-trimethyl-L-lysine (trimethyllysine, TML). Stable-isotope-dilution tandem mass spectrometry analyses of an independent validation cohort (N = 2,140) confirmed TML levels are independently associated with incident (3-year) major adverse cardiovascular event risks (hazards ratio [HR], 2.4; 95% CI, 1.7-3.4) and incident (5-year) mortality risk (HR, 2.9; 95% CI, 2.0-4.2). Genome-wide association studies identified several suggestive loci for TML levels, but none reached genome-wide significance; and d9(trimethyl)-TML isotope tracer studies confirmed TML can serve as a nutrient precursor for gut microbiota-dependent generation of trimethylamine (TMA) and the atherogenic metabolite trimethylamine N-oxide (TMAO). Although TML was shown to be abundant in both plant- and animal-derived foods, mouse and human fecal cultures (omnivores and vegans) showed slow conversion of TML to TMA. Furthermore, unlike chronic dietary choline, TML supplementation in mice failed to elevate plasma TMAO or heighten thrombosis potential in vivo. Thus, TML is identified as a strong predictor of incident CVD risks in subjects and to serve as a dietary precursor for gut microbiota-dependent generation of TMAO; however, TML does not appear to be a major microbial source for TMAO generation in vivo.
Assuntos
Doenças Cardiovasculares/metabolismo , Lisina/análogos & derivados , Metabolômica , Metilaminas/metabolismo , Nutrientes/metabolismo , Idoso , Animais , Aterosclerose/metabolismo , Carnitina , Colesterol/metabolismo , Colina , Modelos Animais de Doenças , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal , Estudo de Associação Genômica Ampla , Humanos , Lisina/sangue , Lisina/genética , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fatores de Risco , TromboseRESUMO
Choline is an essential nutrient and methyl donor required for epigenetic regulation. Here, we assessed the impact of gut microbial choline metabolism on bacterial fitness and host biology by engineering a microbial community that lacks a single choline-utilizing enzyme. Our results indicate that choline-utilizing bacteria compete with the host for this nutrient, significantly impacting plasma and hepatic levels of methyl-donor metabolites and recapitulating biochemical signatures of choline deficiency. Mice harboring high levels of choline-consuming bacteria showed increased susceptibility to metabolic disease in the context of a high-fat diet. Furthermore, bacterially induced reduction of methyl-donor availability influenced global DNA methylation patterns in both adult mice and their offspring and engendered behavioral alterations. Our results reveal an underappreciated effect of bacterial choline metabolism on host metabolism, epigenetics, and behavior. This work suggests that interpersonal differences in microbial metabolism should be considered when determining optimal nutrient intake requirements.
Assuntos
Bactérias/metabolismo , Colina/metabolismo , Epigênese Genética , Intestinos/microbiologia , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Metilação de DNA , Feminino , Microbioma Gastrointestinal , Humanos , Mucosa Intestinal/metabolismo , Masculino , Doenças Metabólicas/microbiologia , Camundongos Endogâmicos C57BLRESUMO
Thrombosis plays an important role in cardiovascular disease (CVD). Platelet activation is an essential step in the genesis and propagation of atherothrombotic complications. In a recent publication, Zhu and colleagues report that gut microbe-derived TMAO enhances platelet responsiveness and thrombosis, providing a novel mechanistic connection between microbes and CVD (Zhu et al., 2016).
Assuntos
Dieta , Trombose , Doenças Cardiovasculares , Humanos , Interações MicrobianasRESUMO
UNLABELLED: Choline is a water-soluble nutrient essential for human life. Gut microbial metabolism of choline results in the production of trimethylamine (TMA), which upon absorption by the host is converted in the liver to trimethylamine-N-oxide (TMAO). Recent studies revealed that TMAO exacerbates atherosclerosis in mice and positively correlates with the severity of this disease in humans. However, which microbes contribute to TMA production in the human gut, the extent to which host factors (e.g., genotype) and diet affect TMA production and colonization of these microbes, and the effects TMA-producing microbes have on the bioavailability of dietary choline remain largely unknown. We screened a collection of 79 sequenced human intestinal isolates encompassing the major phyla found in the human gut and identified nine strains capable of producing TMA from choline in vitro. Gnotobiotic mouse studies showed that TMAO accumulates in the serum of animals colonized with TMA-producing species, but not in the serum of animals colonized with intestinal isolates that do not generate TMA from choline in vitro. Remarkably, low levels of colonization by TMA-producing bacteria significantly reduced choline levels available to the host. This effect was more pronounced as the abundance of TMA-producing bacteria increased. Our findings provide a framework for designing strategies aimed at changing the representation or activity of TMA-producing bacteria in the human gut and suggest that the TMA-producing status of the gut microbiota should be considered when making recommendations about choline intake requirements for humans. IMPORTANCE: Cardiovascular disease (CVD) is the leading cause of death and disability worldwide, and increased trimethylamine N-oxide (TMAO) levels have been causally linked with CVD development. This work identifies members of the human gut microbiota responsible for both the accumulation of trimethylamine (TMA), the precursor of the proatherogenic compound TMAO, and subsequent decreased choline bioavailability to the host. Understanding how to manipulate the representation and function of choline-consuming, TMA-producing species in the intestinal microbiota could potentially lead to novel means for preventing or treating atherosclerosis and choline deficiency-associated diseases.