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1.
Proc Natl Acad Sci U S A ; 118(20)2021 05 18.
Article in English | MEDLINE | ID: mdl-33972416

ABSTRACT

Active inflammatory bowel disease (IBD) often coincides with increases of Ruminococcus gnavus, a gut microbe found in nearly everyone. It was not known how, or if, this correlation contributed to disease. We investigated clinical isolates of R. gnavus to identify molecular mechanisms that would link R. gnavus to inflammation. Here, we show that only some isolates of R. gnavus produce a capsular polysaccharide that promotes a tolerogenic immune response, whereas isolates lacking functional capsule biosynthetic genes elicit robust proinflammatory responses in vitro. Germ-free mice colonized with an isolate of R. gnavus lacking a capsule show increased measures of gut inflammation compared to those colonized with an encapsulated isolate in vivo. These observations in the context of our earlier identification of an inflammatory cell-wall polysaccharide reveal how some strains of R. gnavus could drive the inflammatory responses that characterize IBD.


Subject(s)
Bacterial Capsules/immunology , Clostridiales/immunology , Gastrointestinal Microbiome/immunology , Immunity/immunology , Inflammatory Bowel Diseases/immunology , Polysaccharides/immunology , Adult , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Capsules/chemistry , Bacterial Capsules/ultrastructure , Cells, Cultured , Child , Clostridiales/classification , Clostridiales/genetics , Cytokines/immunology , Cytokines/metabolism , Female , Gastrointestinal Microbiome/drug effects , Gastrointestinal Microbiome/genetics , Humans , Ileum/immunology , Ileum/metabolism , Ileum/microbiology , Inflammatory Bowel Diseases/microbiology , Mice, Inbred C57BL , Multigene Family/genetics , Phylogeny
2.
Nat Chem Biol ; 17(1): 20-29, 2021 01.
Article in English | MEDLINE | ID: mdl-32747812

ABSTRACT

Bariatric surgery, the most effective treatment for obesity and type 2 diabetes, is associated with increased levels of the incretin hormone glucagon-like peptide-1 (GLP-1) and changes in levels of circulating bile acids. The levels of individual bile acids in the gastrointestinal (GI) tract after surgery have, however, remained largely unstudied. Using ultra-high performance liquid chromatography-mass spectrometry-based quantification, we observed an increase in an endogenous bile acid, cholic acid-7-sulfate (CA7S), in the GI tract of both mice and humans after sleeve gastrectomy. We show that CA7S is a Takeda G-protein receptor 5 (TGR5) agonist that increases Tgr5 expression and induces GLP-1 secretion. Furthermore, CA7S administration increases glucose tolerance in insulin-resistant mice in a TGR5-dependent manner. CA7S remains gut restricted, minimizing off-target effects previously observed for TGR5 agonists absorbed into the circulation. By studying changes in individual metabolites after surgery, the present study has revealed a naturally occurring TGR5 agonist that exerts systemic glucoregulatory effects while remaining confined to the gut.


Subject(s)
Anti-Obesity Agents/pharmacology , Bariatric Surgery/methods , Cholic Acid/pharmacology , Obesity/surgery , Receptors, G-Protein-Coupled/genetics , Animals , Anti-Obesity Agents/metabolism , Bile/chemistry , Bile/metabolism , Caco-2 Cells , Cholic Acid/biosynthesis , Colon/metabolism , Gene Expression Regulation , Glucagon-Like Peptide 1/genetics , Glucagon-Like Peptide 1/metabolism , Glucose Tolerance Test , HEK293 Cells , Humans , Insulin Resistance , Male , Mice , Mice, Inbred C57BL , Mice, Obese , Obesity/genetics , Obesity/metabolism , Obesity/pathology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Sulfates
3.
Proc Natl Acad Sci U S A ; 116(26): 12672-12677, 2019 06 25.
Article in English | MEDLINE | ID: mdl-31182571

ABSTRACT

A substantial and increasing number of human diseases are associated with changes in the gut microbiota, and discovering the molecules and mechanisms underlying these associations represents a major research goal. Multiple studies associate Ruminococcus gnavus, a prevalent gut microbe, with Crohn's disease, a major type of inflammatory bowel disease. We have found that R. gnavus synthesizes and secretes a complex glucorhamnan polysaccharide with a rhamnose backbone and glucose sidechains. Chemical and spectroscopic studies indicated that the glucorhamnan was largely a repeating unit of five sugars with a linear backbone formed from three rhamnose units and a short sidechain composed of two glucose units. The rhamnose backbone is made from 1,2- and 1,3-linked rhamnose units, and the sidechain has a terminal glucose linked to a 1,6-glucose. This glucorhamnan potently induces inflammatory cytokine (TNFα) secretion by dendritic cells, and TNFα secretion is dependent on toll-like receptor 4 (TLR4). We also identify a putative biosynthetic gene cluster for this molecule, which has the four biosynthetic genes needed to convert glucose to rhamnose and the five glycosyl transferases needed to build the repeating pentasaccharide unit of the inflammatory glucorhamnan.


Subject(s)
Clostridiales/pathogenicity , Crohn Disease/microbiology , Polysaccharides, Bacterial/toxicity , Animals , Cells, Cultured , Clostridiales/metabolism , Crohn Disease/metabolism , Gastrointestinal Microbiome , Mice , Mice, Inbred C57BL , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/metabolism , Tumor Necrosis Factor-alpha/metabolism
4.
Biochemistry ; 60(34): 2593-2609, 2021 08 31.
Article in English | MEDLINE | ID: mdl-34411482

ABSTRACT

SHP2 is a protein tyrosine phosphatase that plays a critical role in the full activation of the Ras-MAPK pathway upon stimulation of receptor tyrosine kinases, which are frequently amplified or mutationally activated in human cancer. In addition, activating mutations in SHP2 result in developmental disorders and hematologic malignancies. Several allosteric inhibitors have been developed for SHP2 and are currently in clinical trials. Here, we report the development and evaluation of a SHP2 PROTAC created by conjugating RMC-4550 with pomalidomide using a PEG linker. This molecule is highly selective for SHP2, induces degradation of SHP2 in leukemic cells at submicromolar concentrations, inhibits MAPK signaling, and suppresses cancer cell growth. SHP2 PROTACs serve as an alternative strategy for targeting ERK-dependent cancers and are useful tools alongside allosteric inhibitors for dissecting the mechanisms by which SHP2 exerts its oncogenic activity.


Subject(s)
Antineoplastic Agents/pharmacology , Methanol/analogs & derivatives , Neoplasms/drug therapy , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Pyrazines/pharmacology , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Humans , Molecular Targeted Therapy , Mutation , Neoplasms/metabolism , Neoplasms/pathology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Proteolysis , Signal Transduction
5.
J Nat Prod ; 83(3): 744-755, 2020 03 27.
Article in English | MEDLINE | ID: mdl-32105475

ABSTRACT

Over the past 70 years, the search for small molecules from nature has transformed biomedical research: natural products are the basis for half of all pharmaceuticals; the quest for total synthesis of natural products fueled development of methodologies for organic synthesis; and their biosynthesis presented unprecedented biochemical transformations, expanding our chemo-enzymatic toolkit. Initially, the discovery of small molecules was driven by bioactivity-guided fractionation. However, this approach yielded the frequent rediscovery of already known metabolites. As a result, focus shifted to identifying novel scaffolds through either structure-first methods or genome mining, relegating function as a secondary concern. Over the past two decades, the laboratory of Jon Clardy has taken an alternative route and focused on an ecology-driven, function-first approach in pursuit of uncovering bacterial small molecules with biological activity. In this review, we highlight several examples that showcase this ecology-first approach. Though the highlighted systems are diverse, unifying themes are (1) to understand how microbes interact with their host or environment, (2) to gain insights into the environmental roles of microbial metabolites, and (3) to explore pharmaceutical potential from these ecologically relevant metabolites.


Subject(s)
Bacteria/chemistry , Biological Products , Drug Discovery , Biological Products/chemistry , Drug Discovery/methods , Molecular Structure
6.
Nat Chem Biol ; 13(10): 1063-1065, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28805802

ABSTRACT

Despite containing an α-amino acid, the versatile cofactor S-adenosylmethionine (SAM) is not a known building block for nonribosomal peptide synthetase (NRPS) assembly lines. Here we report an unusual NRPS module from colibactin biosynthesis that uses SAM for amide bond formation and subsequent cyclopropanation. Our findings showcase a new use for SAM and reveal a novel biosynthetic route to a functional group that likely mediates colibactin's genotoxicity.


Subject(s)
Cyclopropanes/chemistry , Cyclopropanes/metabolism , Peptide Synthases/metabolism , Peptides/metabolism , Polyketides/metabolism , S-Adenosylmethionine/metabolism , Escherichia coli/metabolism , Peptide Synthases/chemistry , Peptides/chemistry , Polyketides/chemistry , S-Adenosylmethionine/chemistry
7.
Nat Prod Rep ; 33(8): 942-50, 2016 Aug 27.
Article in English | MEDLINE | ID: mdl-27376415

ABSTRACT

Covering: up to 2016In this highlight, we describe the current landscape for dereplication and discovery of natural products based on the measurement of the intact mass by LC-MS. Often it is assumed that because better mass accuracy (provided by higher resolution mass spectrometers) is necessary for absolute chemical formula determination (≤1 part-per-million), that it is also necessary for dereplication of natural products. However, the average ability to dereplicate tapers off at ∼10 ppm, with modest improvement gained from better mass accuracy when querying focused databases of natural products. We also highlight some recent examples of how these platforms are applied to synthetic biology, and recent methods for dereplication and correlation of substructures using tandem MS data. We also offer this highlight to serve as a brief primer for those entering the field of mass spectrometry-based natural products discovery.


Subject(s)
Biological Products/chemistry , Mass Spectrometry/methods , Synthetic Biology , Chromatography, Liquid/methods , Databases, Factual , Molecular Structure , Molecular Weight
8.
Cell Host Microbe ; 31(7): 1075-1076, 2023 07 12.
Article in English | MEDLINE | ID: mdl-37442092

ABSTRACT

New classes of antibiotics are desperately needed in our fight against antibiotic-resistant bacterial infections. Jia et al. publish a new "multi-armed" antibiotic scaffold that effectively treats methicillin-resistant Staphylococcus aureus infections in mice. These compounds are structurally unlike pre-clinical or approved antibiotics, and they may hit an "irresistible" target.


Subject(s)
Methicillin-Resistant Staphylococcus aureus , Staphylococcal Infections , Animals , Mice , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Microbial Sensitivity Tests
9.
mSphere ; 5(5)2020 09 09.
Article in English | MEDLINE | ID: mdl-32907957

ABSTRACT

Multiple epidemiological studies identify Dolosigranulum pigrum as a candidate beneficial bacterium based on its positive association with health, including negative associations with nasal/nasopharyngeal colonization by the pathogenic species Staphylococcus aureus and Streptococcus pneumoniae Using a multipronged approach to gain new insights into D. pigrum function, we observed phenotypic interactions and predictions of genomic capacity that support the idea of a role for microbe-microbe interactions involving D. pigrum in shaping the composition of human nasal microbiota. We identified in vivo community-level and in vitro phenotypic cooperation by specific nasal Corynebacterium species. Also, D. pigrum inhibited S. aureus growth in vitro, whereas robust inhibition of S. pneumoniae required both D. pigrum and a nasal Corynebacterium together. D. pigrum l-lactic acid production was insufficient to account for these inhibitions. Genomic analysis of 11 strains revealed that D. pigrum has a small genome (average 1.86 Mb) and multiple predicted auxotrophies consistent with D. pigrum relying on its human host and on cocolonizing bacteria for key nutrients. Further, the accessory genome of D. pigrum harbored a diverse repertoire of biosynthetic gene clusters, some of which may have a role in microbe-microbe interactions. These new insights into D. pigrum's functions advance the field from compositional analysis to genomic and phenotypic experimentation on a potentially beneficial bacterial resident of the human upper respiratory tract and lay the foundation for future animal and clinical experiments.IMPORTANCEStaphylococcus aureus and Streptococcus pneumoniae infections cause significant morbidity and mortality in humans. For both, nasal colonization is a risk factor for infection. Studies of nasal microbiota identify Dolosigranulum pigrum as a benign bacterium present when adults are free of S. aureus or when children are free of S. pneumoniae Here, we validated these in vivo associations with functional assays. We found that D. pigrum inhibited S. aureusin vitro and, together with a specific nasal Corynebacterium species, also inhibited S. pneumoniae Furthermore, genomic analysis of D. pigrum indicated that it must obtain key nutrients from other nasal bacteria or from humans. These phenotypic interactions support the idea of a role for microbe-microbe interactions in shaping the composition of human nasal microbiota and implicate D. pigrum as a mutualist of humans. These findings support the feasibility of future development of microbe-targeted interventions to reshape nasal microbiota composition to exclude S. aureus and/or S. pneumoniae.


Subject(s)
Bacteria/metabolism , Carnobacteriaceae/physiology , Microbial Interactions , Microbiota , Nasopharynx/microbiology , Bacteria/classification , Bacteria/genetics , Carnobacteriaceae/genetics , Child, Preschool , Genomics , Humans , Infant , Pneumococcal Infections/microbiology , Staphylococcus aureus/genetics , Staphylococcus aureus/physiology , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/physiology
10.
ACS Chem Biol ; 11(8): 2117-23, 2016 08 19.
Article in English | MEDLINE | ID: mdl-27310134

ABSTRACT

Unlocking the biochemical stores of fungi is key for developing future pharmaceuticals. Through reduced expression of a critical histone deacetylase in Aspergillus nidulans, increases of up to 100-fold were observed in the levels of 15 new aspercryptins, recently described lipopeptides with two noncanonical amino acids derived from octanoic and dodecanoic acids. In addition to two NMR-verified structures, MS/MS networking helped uncover an additional 13 aspercryptins. The aspercryptins break the conventional structural orientation of lipopeptides and appear "backward" when compared to known compounds of this class. We have also confirmed the 14-gene aspercryptin biosynthetic gene cluster, which encodes two fatty acid synthases and several enzymes to convert saturated octanoic and dodecanoic acid to α-amino acids.


Subject(s)
Aspergillus nidulans/metabolism , Histone Deacetylases/metabolism , Oligopeptides/metabolism , Aspergillus nidulans/enzymology , Chromatography, Liquid , Metabolomics , Multigene Family , Oligopeptides/biosynthesis , Oligopeptides/genetics , Tandem Mass Spectrometry
11.
ACS Chem Biol ; 10(6): 1535-41, 2015 Jun 19.
Article in English | MEDLINE | ID: mdl-25815712

ABSTRACT

The microbial world offers a rich source of bioactive compounds for those able to sift through it. Technologies capable of quantitatively detecting natural products while simultaneously identifying known compounds would expedite the search for new pharmaceutical leads. Prior efforts have targeted histone deacetylases in fungi to globally activate the production of new secondary metabolites, yet no study has directly assessed its effects with minimal bias at the metabolomic level. Using untargeted metabolomics, we monitored changes in >1000 small molecules secreted from the model fungus, Aspergillus nidulans, following genetic or chemical reductions in histone deacetylase activity (HDACi). Through quantitative, differential analyses, we found that nearly equal numbers of compounds were up- and down-regulated by >100 fold. We detected products from both known and unknown biosynthetic pathways and discovered that A. nidulans is capable of producing fellutamides, proteasome inhibitors whose expression was induced by ∼100 fold or greater upon HDACi. This work adds momentum to an "omics"-driven resurgence in natural products research, where direct detection replaces bioactivity as the primary screen for new pharmacophores.


Subject(s)
Aspergillus nidulans/drug effects , Fungal Proteins/antagonists & inhibitors , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Metabolome/drug effects , Aspergillus nidulans/enzymology , Aspergillus nidulans/genetics , DNA Methylation , Epigenesis, Genetic , Fungal Proteins/genetics , Fungal Proteins/metabolism , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylases/genetics , Lipopeptides/biosynthesis , Lipopeptides/isolation & purification , Metabolome/genetics , Metabolomics , Multigene Family , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism
12.
Science ; 366(6471): 1309-1310, 2019 12 13.
Article in English | MEDLINE | ID: mdl-31831655

Subject(s)
Microbiota , Humans , Metagenome
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