RESUMO
The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis, we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the National Institutes of Health Human Microbiome Project (NIH HMP) stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics, synthetic biology, and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic potential of the microbiota while avoiding the challenges of cultivating fastidious commensals.
Assuntos
Bactérias/metabolismo , Microbioma Gastrointestinal , Microbiota , Peptídeo Sintases/metabolismo , Pirazinas/metabolismo , Animais , Bacillus subtilis/genética , Bactérias/classificação , Bactérias/genética , Escherichia coli/genética , Fezes/microbiologia , Humanos , Peptídeo Sintases/genética , FilogeniaRESUMO
The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of host-microbiota interactions cannot often be recapitulated. We describe the design and development of a novel, versatile and modular in vitro system that enables indirect coculture of human epithelial cells with anaerobic bacteria for the characterization of host-microbe secreted metabolite interactions. This system was designed to compartmentalize anaerobes and human cells in separate chambers conducive to each organism's requisite cell growth conditions. Using perfusion, fluidic mixing, and automated sample collection, the cells continuously received fresh media, while in contact with their corresponding compartments conditioned supernatant. Supernatants from each chamber were collected in a cell-free time-resolved fashion. The system sustained low oxygen conditions in the anaerobic chamber, while also supporting the growth of a representative anaerobe (Bacteroides thetaiotaomicron) and a human colonic epithelial cell line (Caco-2) in the aerobic chamber. Caco-2 global gene expression changes in response to coculture with B. thetaiotaomicron was characterized using RNA sequencing. Extensive, targeted metabolomics analysis of over 150 central carbon metabolites was performed on the serially collected supernatants. We observed broad metabolite changes in host-microbe coculture, compared to respective mono-culture controls. These effects were dependent both on sampling time and the compartment probed (apical vs. basolateral). Coculturing resulted in the depletion of several important metabolites, including guanine, uridine 5'-monophosphate, asparagine, and thiamine. Additionally, while Caco-2 cells cultured alone predominantly affected the basolateral metabolite milieu, increased abundance of 2,3-dihydroxyisovalerate and thymine on the basolateral side, occurred when the cells were cocultured with B. thetaiotaomicron. Thus, our system can capture the dynamic, competitive and cooperative processes between host cells and gut microbes.
Assuntos
Reatores Biológicos , Técnicas de Cocultura , Células Epiteliais , Humanos , Reatores Biológicos/microbiologia , Células Epiteliais/microbiologia , Células Epiteliais/metabolismo , Células CACO-2 , Microbioma Gastrointestinal/fisiologia , Bacteroides thetaiotaomicron/metabolismoRESUMO
Neisseria meningitidis is a Gram-negative opportunistic pathogen that is responsible for causing human diseases with high mortality, such as septicemia and meningitis. The molecular mechanisms N.â meningitidis employ to manipulate the immune system, translocate the mucosal and blood-brain barriers, and exert virulence are largely unknown. Human-associated bacteria encode a variety of bioactive small molecules with growing evidence for N-acyl amides as being important signaling molecules. However, only a small fraction of these metabolites has been identified from the human microbiota thus far. Here, we heterologously expressed an N-acyltransferase encoded in the obligate human pathogen N.â meningitidis and identified 30â N-acyl amides with representative members serving as agonists of the G-protein coupled receptor (GPCR) S1PR4. During this process, we also characterized two mammalian N-acyl amides derived from the bovine medium. Both groups of metabolites suppress anti-inflammatory interleukin-10 signaling in human macrophage cell types, but they also suppress the pro-inflammatory interleukin-17A+ population in TH 17-differentiated CD4+ T cells.
Assuntos
Neisseria meningitidis , Humanos , Bovinos , Animais , Esfingosina , Amidas/farmacologia , Virulência , Transdução de Sinais , MamíferosRESUMO
Chemistry drives many biological interactions between the microbiota and host animals, yet it is often challenging to identify the chemicals involved. This poses a problem, as such small molecules are excellent sources of potential pharmaceuticals, pretested by nature for animal compatibility. We discovered anti-HIV compounds from small, marine tunicates from the Eastern Fields of Papua New Guinea. Tunicates are a reservoir for new bioactive chemicals, yet their small size often impedes identification or even detection of the chemicals within. We solved this problem by combining chemistry, metagenomics, and synthetic biology to directly identify and synthesize the natural products. We show that these anti-HIV compounds, the divamides, are a novel family of lanthipeptides produced by symbiotic bacteria living in the tunicate. Neighboring animal colonies contain structurally related divamides that differ starkly in their biological properties, suggesting a role for biosynthetic plasticity in a native context wherein biological interactions take place.
Assuntos
Fármacos Anti-HIV/farmacologia , Produtos Biológicos/farmacologia , Descoberta de Drogas , Infecções por HIV/tratamento farmacológico , Microbiota , Simbiose , Animais , Bactérias , DNA/análise , Avaliação Pré-Clínica de Medicamentos , Genômica , Humanos , Lisinoalanina/química , Metagenoma , Metagenômica , Família Multigênica , Peptídeos/farmacologia , Relação Estrutura-Atividade , Biologia Sintética , Linfócitos T/efeitos dos fármacos , UrocordadosRESUMO
Tapinarof is a stilbene drug that is used to treat psoriasis and atopic dermatitis, and is thought to function through regulation of the AhR and Nrf2 signaling pathways, which have also been linked to inflammatory bowel diseases. It is produced by the gammaproteobacterial Photorhabdus genus, which thus represents a model to probe tapinarof structural and functional transformations. We show that Photorhabdus transforms tapinarof into novel drug metabolism products that kill inflammatory bacteria, and that a cupin enzyme contributes to the conversion of tapinarof and related dietary stilbenes into novel dimers. One dimer has activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE), and another undergoes spontaneous cyclizations to a cyclopropane-bridge-containing hexacyclic framework that exhibits activity against Mycobacterium. These dimers lack efficacy in a colitis mouse model, whereas the monomer reduces disease symptoms.
Assuntos
Antibacterianos/metabolismo , Autoimunidade/efeitos dos fármacos , Fatores Imunológicos/metabolismo , Photorhabdus/metabolismo , Resorcinóis/metabolismo , Estilbenos/metabolismo , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Biotransformação , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Camundongos , Resorcinóis/química , Resorcinóis/farmacologia , Estilbenos/química , Estilbenos/farmacologiaRESUMO
Steroids can be difficult to modify through traditional organic synthesis methods, but many enzymes regio- and stereoselectively process a wide variety of steroid substrates. We tested whether steroid-modifying enzymes could make novel steroids from non-native substrates. Numerous genes encoding steroid-modifying enzymes, including some bacterial enzymes, were expressed in mammalian cells by transient transfection and found to be active. We made three unusual steroids by stable expression, in HEK293 cells, of the 7α-hydroxylase CYP7B1, which was selected because of its high native product yield. These cells made 7α,17α-dihydroxypregnenolone and 7ß,17α-dihydroxypregnenolone from 17α-hydroxypregnenolone and produced 11α,16α-dihydroxyprogesterone from 16α-hydroxyprogesterone. The last two products were the result of CYP7B1-catalyzed hydroxylation at previously unobserved sites. A Rosetta docking model of CYP7B1 suggested that these substrates' D-ring hydroxy groups might prevent them from binding in the same way as the native substrates, bringing different carbon atoms close to the active ferryl oxygen atom. This new approach could potentially use other enzymes and substrates to produce many novel steroids for drug candidate testing.
Assuntos
Família 7 do Citocromo P450/metabolismo , Esteroide Hidroxilases/metabolismo , Esteroides/biossíntese , Domínio Catalítico , Engenharia Celular/métodos , Família 7 do Citocromo P450/química , Células HEK293 , Humanos , Hidroxilação , Simulação de Acoplamento Molecular , Ligação Proteica , Esteroide Hidroxilases/química , Esteroides/química , Esteroides/metabolismo , Especificidade por SubstratoRESUMO
Fungus-growing ants engage in complex symbiotic relationships with their fungal crop, specialized fungal pathogens, and bacteria that provide chemical defenses. In an effort to understand the evolutionary origins of this multilateral system, we investigated bacteria isolated from fungi. One bacterial strain (Streptomyces sp. CLI2509) from the bracket fungus Hymenochaete rubiginosa, produced an unusual peptide, tryptorubin A, which contains heteroaromatic links between side chains that give it a rigid polycyclic globular structure. The three-dimensional structure was determined by NMR and MS, including a 13C-13C COSY of isotopically enriched material, degradation, derivatives, and computer modeling. Whole genome sequencing identified a likely pair of biosynthetic genes responsible for tryptorubin A's linear hexapeptide backbone. The genome also revealed the close relationship between CLI2509 and Streptomyces sp. SPB78, which was previously implicated in an insect-bacterium symbiosis.
Assuntos
Basidiomycota/química , Peptídeos Cíclicos/biossíntese , Streptomyces/química , Basidiomycota/metabolismo , Conformação Molecular , Peptídeos Cíclicos/química , Streptomyces/metabolismoRESUMO
Utilization of (2)H, (13)C, and (15)N isotopically labeled proteins and peptides is now routine in biomolecular NMR investigations. The widespread availability of inexpensive, uniformly (13) C enriched glucose now makes it possible to isolate uniformly (13)C labeled natural products from microbial fermentation. We now wish to describe an approach for the rapid structural characterization of uniformly (13)C labeled natural products that avoids the pitfalls of relying on parameters typically employed in biomolecular NMR studies.
Assuntos
Algoritmos , Produtos Biológicos/química , Biopolímeros/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Modelos Químicos , Espectroscopia de Prótons por Ressonância Magnética/métodos , Produtos Biológicos/análise , Simulação por Computador , Marcação por Isótopo/métodos , Peso MolecularRESUMO
Utilization of isotopically labeled proteins and peptides is a routinely employed approach in biomolecular NMR investigations. The widespread availability of inexpensive, uniformly (13) C-enriched glucose now makes it possible to produce uniformly (13) C-labeled natural products by microbial fermentation. In this feature article, the authors describe an experimental approach for the rapid structural characterization of uniformly (13) C-labeled natural products based on the Constant-Time HSQC (CT-HSQC) experiment. Rigorous theoretical evaluation of the CT-HSQC experiment allowed the applicability of the experiment to be expanded from the traditional, narrow scope of labeled amino acids to encompass virtually any small molecule or U-(13) C labeled natural product. A suite of experiments including CT-HSQC, (13) C-(13) C COSY, and COSYLR experiments is sufficient for the structure elucidation of uniformly (13) C-labeled small molecules and natural products. Differences in NMR approaches for structure elucidation of natural abundance and uniformly (13) C-labeled molecules are also discussed. The present work provides a researcher working in this area of natural products chemistry with NMR structure elucidation tools for investigating (13) C-labeled small molecules and natural products.
RESUMO
Two novel trialkyl-substituted aromatic acids, solwaric acids A and B, were isolated from a marine Solwaraspora sp. cultivated from the ascidian Trididemnum orbiculatum. Solwaric acids A and B were isotopically labeled with U-¹³C glucose, and analysis of a ¹³C-¹³C COSY allowed for unambiguous determination of the location of the phenyl methyl group. The two novel compounds demonstrated antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA).
Assuntos
Ácidos/farmacologia , Antibacterianos/farmacologia , Urocordados/química , Ácidos/química , Ácidos/isolamento & purificação , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacosRESUMO
Forazolineâ A, a novel antifungal polyketide with inâ vivo efficacy against Candida albicans, was discovered using LCMS-based metabolomics to investigate marine-invertebrate-associated bacteria. Forazolineâ A had a highly unusual and unprecedented skeleton. Acquisition of (13)C-(13)C gCOSY and (13)C-(15)N HMQCâ NMR data provided the direct carbon-carbon and carbon-nitrogen connectivity, respectively. This approach represents the first example of determining direct (13)C-(15)N connectivity for a natural product. Using yeast chemical genomics, we propose that forazolineâ A operated through a new mechanism of action with a phenotypic outcome of disrupting membrane integrity.
Assuntos
Antifúngicos/farmacologia , Bactérias/química , Policetídeos/farmacologia , Animais , Antifúngicos/isolamento & purificação , Candida albicans/efeitos dos fármacos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Espectroscopia de Ressonância Magnética , Biologia Marinha , Espectrometria de Massas , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Policetídeos/isolamento & purificaçãoRESUMO
Type 2 diabetes (T2D) is a global public health issue characterized by excess weight, abdominal obesity, dyslipidemia, hyperglycemia, and a progressive increase in insulin resistance. Human population studies of T2D development and its effects on systemic metabolism are confounded by many factors that cannot be controlled, complicating the interpretation of results and the identification of early biomarkers. Aged, sedentary, and overweight/obese non-human primates (NHPs) are one of the best animal models to mimic spontaneous T2D development in humans. We sought to identify and distinguish a set of plasma and/or fecal metabolite biomarkers, that have earlier disease onset predictability, and that could be evaluated for their predictability in subsequent T2D studies in human cohorts. In this study, a single plasma and fecal sample was collected from each animal in a colony of 57 healthy and dysmetabolic NHPs and analyzed for metabolomics and lipidomics. The samples were comprehensively analyzed using untargeted and targeted LC/MS/MS. The changes in each animal's disease phenotype were monitored using IVGTT, HbA1c, and other clinical metrics, and correlated with their metabolic profile. The plasma and fecal lipids, as well as bile acid profiles, from Healthy, Dysmetabolic (Dys), and Diabetic (Dia) animals were compared. Following univariate and multivariate analyses, including adjustments for weight, age, and sex, several plasma lipid species were identified to be significantly different between these animal groups. Medium and long-chain plasma phosphatidylcholines (PCs) ranked highest at distinguishing Healthy from Dys animals, whereas plasma triglycerides (TG) primarily distinguished Dia from Dys animals. Random Forest (RF) analysis of fecal bile acids showed a reduction in the secondary bile acid glycoconjugate, GCDCA, in diseased animals (AUC 0.76[0.64, 0.89]). Moreover, metagenomics results revealed several bacterial species, belonging to the genera Roseburia, Ruminococcus, Clostridium, and Streptococcus, to be both significantly enriched in non-healthy animals and associated with secondary bile acid levels. In summary, our results highlight the detection of several elevated circulating plasma PCs and microbial species associated with fecal secondary bile acids in NHP dysmetabolic states. The lipids and metabolites we have identified may help researchers to differentiate individual NHPs more precisely between dysmetabolic and overtly diabetic states. This could help assign animals to study groups that are more likely to respond to potential therapies where a difference in efficacy might be anticipated between early vs. advanced disease.
RESUMO
Two novel chlorinated pyrones, halomadurones A and B, and two novel brominated analogues, halomadurones C and D, were isolated from a marine Actinomadura sp. cultivated from the ascidian Ecteinascidia turbinata. Additionally, a non-halogenated analogue, 2-methyl-6-((E)-3-methyl-1,3-hexadiene)-γ-pyrone, was synthesized to understand the role of the halogens for activity. Halomadurones C and D demonstrated potent nuclear factor E2-related factor antioxidant response element (Nrf2-ARE) activation, which is an important therapeutic approach for treatment of neurodegenerative diseases.
Assuntos
Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Animais , Elementos de Resposta Antioxidante/genética , Bactérias/genética , Bactérias/metabolismo , Células Cultivadas , Halogênios/metabolismo , Camundongos , Doenças Neurodegenerativas/tratamento farmacológico , Pironas/metabolismoRESUMO
Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Despite continued efforts to understand the pathophysiology of sepsis, no effective therapies are currently available. While singular components of the aberrant immune response have been investigated, comprehensive studies linking different data layers are lacking. Using an integrated systems immunology approach, we evaluated neutrophil phenotypes and concomitant changes in cytokines and metabolites in patients with sepsis. Our findings identify differentially expressed mature and immature neutrophil subsets in patients with sepsis. These subsets correlate with various proteins, metabolites, and lipids, including pentraxin-3, angiopoietin-2, and lysophosphatidylcholines, in patients with sepsis. These results enabled the construction of a statistical model based on weighted multi-omics linear regression analysis for sepsis biomarker identification. These findings could help inform early patient stratification and treatment options, and facilitate further mechanistic studies targeting the trifecta of surface marker expression, cytokines, and metabolites.
RESUMO
IMPORTANCE: We show that simultaneous study of stool and nasopharyngeal microbiome reveals divergent timing and patterns of maturation, suggesting that local mucosal factors may influence microbiome composition in the gut and respiratory system. Antibiotic exposure in early life as occurs commonly, may have an adverse effect on vaccine responsiveness. Abundance of gut and/or nasopharyngeal bacteria with the machinery to produce lipopolysaccharide-a toll-like receptor 4 agonist-may positively affect future vaccine protection, potentially by acting as a natural adjuvant. The increased levels of serum phenylpyruvic acid in infants with lower vaccine-induced antibody levels suggest an increased abundance of hydrogen peroxide, leading to more oxidative stress in low vaccine-responding infants.
Assuntos
Microbioma Gastrointestinal , Microbiota , Vacinas , Lactente , Criança , Humanos , Metaboloma , VacinaçãoRESUMO
Natural products profoundly impact many research areas, including medicine, organic chemistry, and cell biology. However, discovery of new natural products suffers from a lack of high throughput analytical techniques capable of identifying structural novelty in the face of a high degree of chemical redundancy. Methods to select bacterial strains for drug discovery have historically been based on phenotypic qualities or genetic differences and have not been based on laboratory production of secondary metabolites. Therefore, untargeted LC/MS-based secondary metabolomics was evaluated to rapidly and efficiently analyze marine-derived bacterial natural products using LC/MS-principal component analysis (PCA). A major goal of this work was to demonstrate that LC/MS-PCA was effective for strain prioritization in a drug discovery program. As proof of concept, we evaluated LC/MS-PCA for strain selection to support drug discovery, for the discovery of unique natural products, and for rapid assessment of regulation of natural product production.
Assuntos
Produtos Biológicos/análise , Metabolômica , Bactérias/metabolismo , Produtos Biológicos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Análise de Componente Principal , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A marine Nocardia sp. isolated from the ascidian Trididemnum orbiculatum was found to produce five new lipopeptides, peptidolipins B-F (1-5), which show distinct similarities to the previously reported L-Val(6) analog of peptidolipin NA. Synthetic modification of peptidolipin E (4) was used to determine the location of an olefin within the lipid chain. The advanced Marfey's method was used to determine the absolute configurations of the amino acids. Peptidolipins B (1) and E (4) demonstrated moderate antibacterial activity against methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus.
Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Lipopeptídeos/isolamento & purificação , Lipopeptídeos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Nocardia/química , Urocordados/microbiologia , Animais , Antibacterianos/química , Lipopeptídeos/química , Biologia Marinha , Testes de Sensibilidade Microbiana , Estrutura MolecularRESUMO
A marine Verrucosispora sp. isolated from the sponge Chondrilla caribensis f. caribensis was found to produce thiocoraline, a potent cytotoxic compound. Five new analogs of thiocoraline were isolated and represent the first analogs of thiocoraline. 22'-Deoxythiocoraline (2), thiochondrilline C (5), and 12'-sulfoxythiocoraline (6) demonstrated significant cytotoxicity against the A549 human cancer cell line with EC(50) values of 0.13, 2.86, and 1.26 µM, respectively. The analogs provide insight into the SAR and biosynthesis of thiocoraline. The DP4 probability method was used to analyze ab initio NMR calculations to confirm stereochemical assignments.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Depsipeptídeos/biossíntese , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Micromonosporaceae/metabolismo , Animais , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , EstereoisomerismoRESUMO
We recently developed a 2D 1H-13C HSQC0 approach to quantify individual chemicals in complex mixtures. The HSQC0 approach has been implemented in phase-cycled and gradient-selective versions. As in quantitative 1D NMR, the normalized integrated signal intensities in HSQC0 are proportional to the concentrations of individual chemicals in the mixture. We applied the HSQC0 approach to selectively quantify thiocoraline present at a level of 1% w/w in an extract from a Verrucosispora sp. isolated from the sponge Chondrilla caribensis f. caribensis. We expect that this approach can be used to quantify other natural products of interest in extracts without prior purification.
Assuntos
Depsipeptídeos/análise , Micromonosporaceae/química , Poríferos/microbiologia , Animais , Depsipeptídeos/química , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
Donor-to-donor variability in primary human organoid cultures has not been well characterized. As these cultures contain multiple cell types, there is greater concern that variability could lead to increased noise. In this work we investigated donor-to-donor variability in human gut adult stem cell (ASC) organoids. We examined intestinal developmental pathways during culture differentiation in ileum- and colon-derived cultures established from multiple donors, showing that differentiation patterns were consistent among cultures. This finding indicates that donor-to-donor variability in this system remains at a manageable level. Intestinal metabolic activity was evaluated by targeted analysis of central carbon metabolites and by analyzing hormone production patterns. Both experiments demonstrated similar metabolic functions among donors. Importantly, this activity reflected intestinal biology, indicating that these ASC organoid cultures are appropriate for studying metabolic processes. This work establishes a framework for generating high-confidence data using human primary cultures through thorough characterization of variability.