RESUMEN
Group 3 innate lymphoid cells (ILC3s) sense environmental signals that are critical for gut homeostasis and host defense. However, the metabolite-sensing G-protein-coupled receptors that regulate colonic ILC3s remain poorly understood. We found that colonic ILC3s expressed Ffar2, a microbial metabolite-sensing receptor, and that Ffar2 agonism promoted ILC3 expansion and function. Deficiency of Ffar2 in ILC3s decreased their in situ proliferation and ILC3-derived interleukin-22 (IL-22) production. This led to impaired gut epithelial function characterized by altered mucus-associated proteins and antimicrobial peptides and increased susceptibility to colonic injury and bacterial infection. Ffar2 increased IL-22+ CCR6+ ILC3s and influenced ILC3 abundance in colonic lymphoid tissues. Ffar2 agonism differentially activated AKT or ERK signaling and increased ILC3-derived IL-22 via an AKT and STAT3 axis. Our findings suggest that Ffar2 regulates colonic ILC3 proliferation and function, and they identify an ILC3-receptor signaling pathway modulating gut homeostasis and pathogen defense.
Asunto(s)
Inmunidad Innata , Inmunidad Mucosa , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Biomarcadores , Citocinas/metabolismo , Susceptibilidad a Enfermedades , Microbioma Gastrointestinal/inmunología , Expresión Génica , Humanos , Inmunomodulación , Mucosa Intestinal/patología , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt , Receptores de Superficie Celular/agonistas , Factor de Transcripción STAT3/metabolismoRESUMEN
Microbial communities and their associated bioactive compounds1-3 are often disrupted in conditions such as the inflammatory bowel diseases (IBD)4. However, even in well-characterized environments (for example, the human gastrointestinal tract), more than one-third of microbial proteins are uncharacterized and often expected to be bioactive5-7. Here we systematically identified more than 340,000 protein families as potentially bioactive with respect to gut inflammation during IBD, about half of which have not to our knowledge been functionally characterized previously on the basis of homology or experiment. To validate prioritized microbial proteins, we used a combination of metagenomics, metatranscriptomics and metaproteomics to provide evidence of bioactivity for a subset of proteins that are involved in host and microbial cell-cell communication in the microbiome; for example, proteins associated with adherence or invasion processes, and extracellular von Willebrand-like factors. Predictions from high-throughput data were validated using targeted experiments that revealed the differential immunogenicity of prioritized Enterobacteriaceae pilins and the contribution of homologues of von Willebrand factors to the formation of Bacteroides biofilms in a manner dependent on mucin levels. This methodology, which we term MetaWIBELE (workflow to identify novel bioactive elements in the microbiome), is generalizable to other environmental communities and human phenotypes. The prioritized results provide thousands of candidate microbial proteins that are likely to interact with the host immune system in IBD, thus expanding our understanding of potentially bioactive gene products in chronic disease states and offering a rational compendium of possible therapeutic compounds and targets.
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Proteínas Bacterianas , Microbioma Gastrointestinal , Genes Microbianos , Enfermedades Inflamatorias del Intestino , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Enfermedad Crónica , Microbioma Gastrointestinal/genética , Humanos , Enfermedades Inflamatorias del Intestino/microbiología , Metagenómica , Proteómica , Reproducibilidad de los Resultados , TranscriptomaRESUMEN
The microbiota modulates gut immune homeostasis. Bacteria influence the development and function of host immune cells, including T helper cells expressing interleukin-17A (TH17 cells). We previously reported that the bile acid metabolite 3-oxolithocholic acid (3-oxoLCA) inhibits TH17 cell differentiation1. Although it was suggested that gut-residing bacteria produce 3-oxoLCA, the identity of such bacteria was unknown, and it was unclear whether 3-oxoLCA and other immunomodulatory bile acids are associated with inflammatory pathologies in humans. Here we identify human gut bacteria and corresponding enzymes that convert the secondary bile acid lithocholic acid into 3-oxoLCA as well as the abundant gut metabolite isolithocholic acid (isoLCA). Similar to 3-oxoLCA, isoLCA suppressed TH17 cell differentiation by inhibiting retinoic acid receptor-related orphan nuclear receptor-γt, a key TH17-cell-promoting transcription factor. The levels of both 3-oxoLCA and isoLCA and the 3α-hydroxysteroid dehydrogenase genes that are required for their biosynthesis were significantly reduced in patients with inflammatory bowel disease. Moreover, the levels of these bile acids were inversely correlated with the expression of TH17-cell-associated genes. Overall, our data suggest that bacterially produced bile acids inhibit TH17 cell function, an activity that may be relevant to the pathophysiology of inflammatory disorders such as inflammatory bowel disease.
Asunto(s)
Bacterias , Ácidos y Sales Biliares , Enfermedades Inflamatorias del Intestino , Bacterias/metabolismo , Diferenciación Celular , Tracto Gastrointestinal/microbiología , Humanos , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/microbiología , Interleucina-17 , Ácido Litocólico/metabolismo , Ácido Litocólico/farmacología , Células Th17RESUMEN
A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1-9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11-13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.
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Ácidos y Sales Biliares/biosíntesis , Ácidos y Sales Biliares/química , Metabolómica , Microbiota/fisiología , Animales , Ácidos y Sales Biliares/metabolismo , Ácido Cólico/biosíntesis , Ácido Cólico/química , Ácido Cólico/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Vida Libre de Gérmenes , Humanos , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/microbiología , Ratones , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
Microbial biochemistry is central to the pathophysiology of inflammatory bowel diseases (IBD). Improved knowledge of microbial metabolites and their immunomodulatory roles is thus necessary for diagnosis and management. Here, we systematically analyzed the chemical, ecological, and epidemiological properties of ~82k metabolic features in 546 Integrative Human Microbiome Project (iHMP/HMP2) metabolomes, using a newly developed methodology for bioactive compound prioritization from microbial communities. This suggested >1000 metabolic features as potentially bioactive in IBD and associated ~43% of prevalent, unannotated features with at least one well-characterized metabolite, thereby providing initial information for further characterization of a significant portion of the fecal metabolome. Prioritized features included known IBD-linked chemical families such as bile acids and short-chain fatty acids, and less-explored bilirubin, polyamine, and vitamin derivatives, and other microbial products. One of these, nicotinamide riboside, reduced colitis scores in DSS-treated mice. The method, MACARRoN, is generalizable with the potential to improve microbial community characterization and provide therapeutic candidates.
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Colitis , Enfermedades Inflamatorias del Intestino , Humanos , Animales , Ratones , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/metabolismo , Metaboloma , Ácidos y Sales BiliaresRESUMEN
BACKGROUND & AIMS: Intestinal microbes and their metabolites affect the development of colorectal cancer (CRC). Short-chain fatty acids are metabolites generated by intestinal microbes from dietary fiber. We investigated the mechanisms by which free fatty acid receptor 2 (FFAR2), a receptor for short-chain fatty acids that can affect the composition of the intestinal microbiome, contributes to the pathogenesis of CRC. METHODS: We performed studies with ApcMin/+ mice, ApcMin/+Ffar2-/- mice, mice with conditional disruption of Ffar2 in dendritic cells (DCs) (Ffar2fl/flCD11c-Cre mice), ApcMin/+Ffar2fl/flCD11c-Cre mice, and Ffar2fl/fl mice (controls); some mice were given dextran sodium sulfate to induce colitis, with or without a FFAR2 agonist or an antibody against interleukin 27 (IL27). Colon and tumor tissues were analyzed by histology, quantitative polymerase chain reaction, and 16S ribosomal RNA gene sequencing; lamina propria and mesenteric lymph node tissues were analyzed by RNA sequencing and flow cytometry. Intestinal permeability was measured after gavage with fluorescently labeled dextran. We collected data on colorectal tumors from The Cancer Genome Atlas. RESULTS: ApcMin/+Ffar2-/- mice developed significantly more spontaneous colon tumors than ApcMin/+ mice and had increased gut permeability before tumor development, associated with reduced expression of E-cadherin. Colon tumors from ApcMin/+Ffar2-/- mice had a higher number of bacteria than tumors from ApcMin/+ mice, as well as higher frequencies of CD39+CD8+ T cells and exhausted or dying T cells. DCs from ApcMin/+Ffar2-/- mice had an altered state of activation, increased death, and higher production of IL27. Administration of an antibody against IL27 reduced the numbers of colon tumors in ApcMin/+ mice with colitis. Frequencies of CD39+CD8+ T cells and IL27+ DCs were increased in colon lamina propria from Ffar2fl/flCD11c-Cre mice with colitis compared with control mice or mice without colitis. ApcMin/+Ffar2fl/flCD11c-Cre mice developed even more tumors than ApcMin/+Ffar2fl/fl mice, and their tumors had even higher numbers of IL27+ DCs. ApcMin/+ mice with colitis given the FFAR2 agonist developed fewer colon tumors, with fewer IL27+ DCs, than mice not given the agonist. DCs incubated with the FFAR2 agonist no longer had gene expression patterns associated with activation or IL27 production. CONCLUSIONS: Loss of FFAR2 promotes colon tumorigenesis in mice by reducing gut barrier integrity, increasing tumor bacterial load, promoting exhaustion of CD8+ T cells, and overactivating DCs, leading to their death. Antibodies against IL27 and an FFAR2 agonist reduce tumorigenesis in mice and might be developed for the treatment of CRC.
Asunto(s)
Colitis/patología , Neoplasias del Colon/inmunología , Células Dendríticas/inmunología , Microbioma Gastrointestinal/inmunología , Interleucinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/inmunología , Colitis/inducido químicamente , Colitis/inmunología , Colon/efectos de los fármacos , Colon/microbiología , Colon/patología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Células Dendríticas/metabolismo , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ácidos Grasos no Esterificados/metabolismo , Femenino , Humanos , Interleucinas/inmunología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Noqueados , Permeabilidad , Cultivo Primario de Células , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genéticaRESUMEN
A major roadblock to understanding how microbes in the gastrointestinal tract colonize and influence the physiology of their hosts is our inability to genetically manipulate new bacterial species and experimentally assess the function of their genes. We describe the application of population-based genomic sequencing after chemical mutagenesis to map bacterial genes responsible for motility in Exiguobacterium acetylicum, a representative intestinal Firmicutes bacterium that is intractable to molecular genetic manipulation. We derived strong associations between mutations in 57 E. acetylicum genes and impaired motility. Surprisingly, less than half of these genes were annotated as motility-related based on sequence homologies. We confirmed the genetic link between individual mutations and loss of motility for several of these genes by performing a large-scale analysis of spontaneous suppressor mutations. In the process, we reannotated genes belonging to a broad family of diguanylate cyclases and phosphodiesterases to highlight their specific role in motility and assigned functions to uncharacterized genes. Furthermore, we generated isogenic strains that allowed us to establish that Exiguobacterium motility is important for the colonization of its vertebrate host. These results indicate that genetic dissection of a complex trait, functional annotation of new genes, and the generation of mutant strains to define the role of genes in complex environments can be accomplished in bacteria without the development of species-specific molecular genetic tools.
Asunto(s)
Firmicutes/genética , Técnicas Genéticas , Animales , Tracto Gastrointestinal/microbiología , Genes Bacterianos , Proteínas Motoras Moleculares/genética , Mutagénesis , Pez CebraRESUMEN
Pathogenic bacteria such as Listeria and Yersinia gain initial entry by binding to host target cells and stimulating their internalization. Bacterial uptake entails successive, increasingly strong associations between receptors on the surface of bacteria and hosts. Even with genetically identical cells grown in the same environment, there are vast differences in the number of bacteria entering any given cell. To gain insight into this variability, we examined uptake dynamics of Escherichia coli engineered to express the invasin surface receptor from Yersinia, which enables uptake via mammalian host ß1-integrins. Surprisingly, we found that the uptake probability of a single bacterium follows a simple power-law dependence on the concentration of integrins. Furthermore, the value of a power-law parameter depends on the particular host-bacterium pair but not on bacterial concentration. This power-law captures the complex, variable processes underlying bacterial invasion while also enabling differentiation of cell lines.
Asunto(s)
Bacterias/metabolismo , Bacterias/patogenicidad , Interacciones Huésped-Patógeno/fisiología , Proteínas de la Membrana/metabolismo , Adhesión Bacteriana , Infecciones Bacterianas , Proteínas Bacterianas/metabolismo , Biología Computacional , Células HeLa , Humanos , Modelos BiológicosRESUMEN
Alterations in intestinal permeability and the gut microbiome caused by alcohol abuse are associated with alcoholic liver disease and with worsening of inflammatory bowel diseases (IBD) symptoms. To resolve the direct effects of chronic ethanol consumption on the colon and its microbiome in the absence of acute or chronic alcohol-induced liver disease, we developed a mouse model of chronic binge drinking that uncovers how alcohol may enhance susceptibility to colitis via the microbiota. Employing daily ethanol gavage, we recapitulate key features of binge ethanol consumption. We found that binge ethanol drinking worsens intestinal infection, colonic injury and inflammation, and this effect persists beyond the drinking period. Using gnotobiotics, we showed that alcohol-driven susceptibility to colitis is microbiota-dependent and transferable to ethanol-naïve mice by microbiome transplantation. Allobaculum spp. expanded in binge drinking mice, and administration of Allobaculum fili was sufficient to enhance colitis in non-drinking mice. Our study provides a model to study binge drinking-microbiota interactions and their effects on host disease and reinforces the pathogenic function of Allobaculum spp. as colitogenic bacteria. Our findings illustrate how chronic binge drinking-induced alterations of the microbiome may affect susceptibility to IBD onset or flares.
Asunto(s)
Consumo Excesivo de Bebidas Alcohólicas , Colitis , Colon , Microbioma Gastrointestinal , Ratones Endogámicos C57BL , Animales , Consumo Excesivo de Bebidas Alcohólicas/complicaciones , Microbioma Gastrointestinal/efectos de los fármacos , Ratones , Colitis/microbiología , Colitis/inducido químicamente , Colon/microbiología , Colon/patología , Modelos Animales de Enfermedad , Bacterias/clasificación , Bacterias/aislamiento & purificación , Bacterias/genética , Etanol/efectos adversos , Susceptibilidad a Enfermedades , Masculino , Vida Libre de Gérmenes , Inflamación/microbiología , Enfermedades Inflamatorias del Intestino/microbiología , Enfermedades Inflamatorias del Intestino/patologíaRESUMEN
Drug platforms that enable the directed delivery of therapeutics to sites of diseases to maximize efficacy and limit off-target effects are needed. Here, we report the development of PROT3EcT, a suite of commensal Escherichia coli engineered to secrete proteins directly into their surroundings. These bacteria consist of three modular components: a modified bacterial protein secretion system, the associated regulatable transcriptional activator, and a secreted therapeutic payload. PROT3EcT secrete functional single-domain antibodies, nanobodies (Nbs), and stably colonize and maintain an active secretion system within the intestines of mice. Furthermore, a single prophylactic dose of a variant of PROT3EcT that secretes a tumor necrosis factor-alpha (TNF-α)-neutralizing Nb is sufficient to ablate pro-inflammatory TNF levels and prevent the development of injury and inflammation in a chemically induced model of colitis. This work lays the foundation for developing PROT3EcT as a platform for the treatment of gastrointestinal-based diseases.
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Colitis , Anticuerpos de Dominio Único , Animales , Ratones , Escherichia coli , Colitis/inducido químicamente , Colitis/terapia , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
For decades, variability in clinical efficacy of the widely used inflammatory bowel disease (IBD) drug 5-aminosalicylic acid (5-ASA) has been attributed, in part, to its acetylation and inactivation by gut microbes. Identification of the responsible microbes and enzyme(s), however, has proved elusive. To uncover the source of this metabolism, we developed a multi-omics workflow combining gut microbiome metagenomics, metatranscriptomics and metabolomics from the longitudinal IBDMDB cohort of 132 controls and patients with IBD. This associated 12 previously uncharacterized microbial acetyltransferases with 5-ASA inactivation, belonging to two protein superfamilies: thiolases and acyl-CoA N-acyltransferases. In vitro characterization of representatives from both families confirmed the ability of these enzymes to acetylate 5-ASA. A cross-sectional analysis within the discovery cohort and subsequent prospective validation within the independent SPARC IBD cohort (n = 208) found three of these microbial thiolases and one acyl-CoA N-acyltransferase to be epidemiologically associated with an increased risk of treatment failure among 5-ASA users. Together, these data address a longstanding challenge in IBD management, outline a method for the discovery of previously uncharacterized gut microbial activities and advance the possibility of microbiome-based personalized medicine.
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Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino , Humanos , Mesalamina/uso terapéutico , Microbioma Gastrointestinal/genética , Estudios Transversales , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Resultado del TratamientoRESUMEN
Members of the human gut microbiome enzymatically process many bioactive molecules in the gastrointestinal tract. Most gut bacterial modifications characterized so far are hydrolytic or reductive in nature. Here we report that abundant human gut bacteria from the phylum Bacteroidetes perform conjugative modifications by selectively sulfonating steroidal metabolites. While sulfonation is a ubiquitous biochemical modification, this activity has not yet been characterized in gut microbes. Using genetic and biochemical approaches, we identify a widespread biosynthetic gene cluster that encodes both a sulfotransferase (BtSULT, BT0416) and enzymes that synthesize the sulfonate donor adenosine 3'-phosphate-5'-phosphosulfate (PAPS), including an APS kinase (CysC, BT0413) and an ATP sulfurylase (CysD and CysN, BT0414-BT0415). BtSULT selectively sulfonates steroidal metabolites with a flat A/B ring fusion, including cholesterol. Germ-free mice monocolonized with Bacteroides thetaiotaomicron ΔBT0416 exhibited reduced gastrointestinal levels of cholesterol sulfate (Ch-S) compared with wild-type B. thetaiotaomicron-colonized mice. The presence of BtSULT and BtSULT homologues in bacteria inhibited leucocyte migration in vitro and in vivo, and abundances of cluster genes were significantly reduced in patients with inflammatory bowel disease. Together, these data provide a mechanism by which gut bacteria sulfonate steroidal metabolites and suggest that these compounds can modulate immune cell trafficking in the host.
Asunto(s)
Bacteroides thetaiotaomicron , Vías Biosintéticas , Animales , Bacterias , Tracto Gastrointestinal , Humanos , Ratones , Sulfato AdenililtransferasaRESUMEN
The intestinal epithelium plays critical roles in sensing and integrating dietary and microbial signals. How microbiota and intestinal epithelial cell (IEC) interactions regulate host physiology in the proximal small intestine, particularly the duodenum, is unclear. Using single-cell RNA sequencing of duodenal IECs under germ-free (GF) and different conventional microbiota compositions, we show that specific microbiota members alter epithelial homeostasis by increasing epithelial turnover rate, crypt proliferation, and major histocompatibility complex class II (MHCII) expression. Microbiome profiling identified Faecalibaculum rodentium as a key species involved in this regulation. F. rodentium decreases enterocyte expression of retinoic-acid-producing enzymes Adh1, Aldh1a1, and Rdh7, reducing retinoic acid signaling required to maintain certain intestinal eosinophil populations. Eosinophils suppress intraepithelial-lymphocyte-mediated production of interferon-γ that regulates epithelial cell function. Thus, we identify a retinoic acid-eosinophil-interferon-γ-dependent circuit by which the microbiota modulates duodenal epithelial homeostasis.
Asunto(s)
Eosinófilos , Tretinoina , Citrobacter rodentium , Células Epiteliales/metabolismo , Firmicutes , Homeostasis , Interferón gamma/metabolismo , Mucosa Intestinal/metabolismo , Tretinoina/metabolismoRESUMEN
The colorectal cancer (CRC)-associated microbiota creates a pro-tumorigenic intestinal milieu and shapes immune responses within the tumor microenvironment. However, how oncomicrobes - like Fusobacterium nucleatum, found in the oral cavity and associated with CRC tissues- affect these distinct aspects of tumorigenesis is difficult to parse. Herein, we found that neonatal inoculation of ApcMin/+ mice with F. nucleatum strain Fn7-1 circumvents technical barriers preventing its intestinal colonization, drives colonic Il17a expression prior to tumor formation, and potentiates intestinal tumorigenesis. Using gnotobiotic mice colonized with a minimal complexity microbiota (the altered Schaedler's flora), we observed that intestinal Fn7-1 colonization increases colonic Th17 cell frequency and their IL-17A and IL-17F expression, along with a concurrent increase in colonic lamina propria Il23p19 expression. As Fn7-1 stably colonizes the intestinal tract in our models, we posited that microbial metabolites, specifically short-chain fatty acids (SCFA) that F. nucleatum abundantly produces in culture and, as we demonstrate, in the intestinal tract, might mediate part of its immunomodulatory effects in vivo. Supporting this hypothesis, we found that Fn7-1 did not alter RORγt+ CD4+T cell frequency in the absence of the SCFA receptor FFAR2. Taken together, our work suggests that F. nucleatum influences intestinal immunity by shaping Th17 responses in an FFAR2-dependent manner, although further studies are necessary to clarify the precise and multifaceted roles of FFAR2. The potential to increase intestinal Th17 responses is shared by another oncomicrobe, enterotoxigenic Bacteroides fragilis, highlighting a conserved pathway that could potentially be targeted to slow oncomicrobe-mediated CRC.
Asunto(s)
Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/microbiología , Fusobacterium nucleatum/fisiología , Interleucina-17/inmunología , Mucosa Intestinal/inmunología , Células Th17/inmunología , Animales , Colon/inmunología , Colon/microbiología , Neoplasias Colorrectales/genética , Femenino , Fusobacterium nucleatum/crecimiento & desarrollo , Microbioma Gastrointestinal , Humanos , Interleucina-17/genética , Mucosa Intestinal/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunologíaRESUMEN
BACKGROUND: High-throughput sequencing provides a powerful window into the structural and functional profiling of microbial communities, but it is unable to characterize only the viable portion of microbial communities at scale. There is as yet not one best solution to this problem. Previous studies have established viability assessments using propidium monoazide (PMA) treatment coupled with downstream molecular profiling (e.g., qPCR or sequencing). While these studies have met with moderate success, most of them focused on the resulting "viable" communities without systematic evaluations of the technique. Here, we present our work to rigorously benchmark "PMA-seq" (PMA treatment followed by 16S rRNA gene amplicon sequencing) for viability assessment in synthetic and realistic microbial communities. RESULTS: PMA-seq was able to successfully reconstruct simple synthetic communities comprising viable/heat-killed Escherichia coli and Streptococcus sanguinis. However, in realistically complex communities (computer screens, computer mice, soil, and human saliva) with E. coli spike-in controls, PMA-seq did not accurately quantify viability (even relative to variability in amplicon sequencing), with its performance largely affected by community properties such as initial biomass, sample types, and compositional diversity. We then applied this technique to environmental swabs from the Boston subway system. Several taxa differed significantly after PMA treatment, while not all microorganisms responded consistently. To elucidate the "PMA-responsive" microbes, we compared our results with previous PMA-based studies and found that PMA responsiveness varied widely when microbes were sourced from different ecosystems but were reproducible within similar environments across studies. CONCLUSIONS: This study provides a comprehensive evaluation of PMA-seq exploring its quantitative potential in synthetic and complex microbial communities, where the technique was effective for semi-quantitative purposes in simple synthetic communities but provided only qualitative assessments in realistically complex community samples. Video abstract.
Asunto(s)
Azidas , Escherichia coli/genética , Viabilidad Microbiana/genética , Microbiota , Propidio/análogos & derivados , Análisis de Secuencia de ADN/métodos , Streptococcus sanguis/genética , Animales , ADN Bacteriano/análisis , ADN Bacteriano/genética , Escherichia coli/aislamiento & purificación , Humanos , Ratones , Microbiota/genética , ARN Ribosómico 16S/genética , Streptococcus sanguis/aislamiento & purificaciónRESUMEN
Bile acids act as signaling molecules that regulate immune homeostasis, including the differentiation of CD4+ T cells into distinct T cell subsets. The bile acid metabolite isoallolithocholic acid (isoalloLCA) enhances the differentiation of anti-inflammatory regulatory T cells (Treg cells) by facilitating the formation of a permissive chromatin structure in the promoter region of the transcription factor forkhead box P3 (Foxp3). Here, we identify gut bacteria that synthesize isoalloLCA from 3-oxolithocholic acid and uncover a gene cluster responsible for the conversion in members of the abundant human gut bacterial phylum Bacteroidetes. We also show that the nuclear hormone receptor NR4A1 is required for the effect of isoalloLCA on Treg cells. Moreover, the levels of isoalloLCA and its biosynthetic genes are significantly reduced in patients with inflammatory bowel diseases, suggesting that isoalloLCA and its bacterial producers may play a critical role in maintaining immune homeostasis in humans.