Publications by authors named "Jason M Crawford"

76 Publications

A Conserved Nonribosomal Peptide Synthetase in Produces Citrulline-Functionalized Lipopeptides.

J Nat Prod 2021 Oct 28;84(10):2692-2699. Epub 2021 Sep 28.

Department of Chemistry, Yale University, New Haven, Connecticut 06511, United States.

The entomopathogenic bacterium exists in a mutualistic relationship with nematodes of the genus . Free-living infective juveniles of prey on insect larvae and regurgitate within the hemocoel of a host larva. subsequently produces a complex array of specialized metabolites and effector proteins that kill the insect and regulate various aspects of the trilateral symbiosis. While species are rich producers of secondary metabolites, many of their biosynthetic gene clusters remain uncharacterized. Here, we describe a nonribosomal peptide synthetase (NRPS) identified through comparative genomics analysis that is widely conserved in species. Heterologous expression of this NRPS gene from in led to the discovery of a family of lipo-tripeptides that chromatographically appear as pairs, containing either a C-terminal carboxylic acid or carboxamide. Coexpression of the NRPS with the leupeptin protease inhibitor pathway enhanced production, facilitating isolation and characterization efforts. The new lipo-tripeptides were also detected in wild-type cultures. These metabolites, termed bovienimides, share an uncommon C-terminal d-citrulline residue. The NRPS lacked a dedicated chain termination domain, resulting in product diversification and release from the assembly line through reactions with ammonia, water, or exogenous alcohols.
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http://dx.doi.org/10.1021/acs.jnatprod.1c00573DOI Listing
October 2021

Escherichia coli small molecule metabolism at the host-microorganism interface.

Nat Chem Biol 2021 10 22;17(10):1016-1026. Epub 2021 Sep 22.

Department of Chemistry, Yale University, New Haven, CT, USA.

Escherichia coli are a common component of the human microbiota, and isolates exhibit probiotic, commensal and pathogenic roles in the host. E. coli members often use diverse small molecule chemistry to regulate intrabacterial, intermicrobial and host-bacterial interactions. While E. coli are considered to be a well-studied model organism in biology, much of their chemical arsenal has only more recently been defined, and much remains to be explored. Here we describe chemical signaling systems in E. coli in the context of the broader field of metabolism at the host-bacteria interface and the role of this signaling in disease modulation.
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http://dx.doi.org/10.1038/s41589-021-00807-5DOI Listing
October 2021

Natural Products: An Era of Discovery in Organic Chemistry.

J Org Chem 2021 08;86(16):10943-10945

Yale University Department of Chemistry New Haven, Connecticut 06520, United States.

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http://dx.doi.org/10.1021/acs.joc.1c01753DOI Listing
August 2021

-Derived γ-Lactams and Structurally Related Metabolites Are Produced at the Intersection of Colibactin and Fatty Acid Biosynthesis.

Org Lett 2021 09 18;23(17):6895-6899. Epub 2021 Aug 18.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

Colibactin is a genotoxic hybrid polyketide-nonribosomal peptide that drives colorectal cancer initiation. While clinical data suggest colibactin genotoxicity is largely caused by the major DNA-cross-linking metabolite, the colibactin locus produces a diverse collection of metabolites with mostly unknown biological activities. Here, we describe 10 new colibactin pathway metabolites () that are dependent on its α-aminomalonyl-carrier protein. The most abundant metabolites, and , were isolated and structurally characterized mainly by nuclear magnetic resonance spectroscopy to be γ-lactam derivatives, and the remaining related structures were inferred via shared biosynthetic logic. Our proposed formation of , which is supported by stereochemical analysis, invokes cross-talk between colibactin and fatty acid biosynthesis, illuminating further the complexity of this diversity-oriented pathway.
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http://dx.doi.org/10.1021/acs.orglett.1c02461DOI Listing
September 2021

Molecules from the Microbiome.

Annu Rev Biochem 2021 06 26;90:789-815. Epub 2021 Mar 26.

Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut 06536, USA; email:

The human microbiome encodes a second genome that dwarfs the genetic capacity of the host. Microbiota-derived small molecules can directly target human cells and their receptors or indirectly modulate host responses through functional interactions with other microbes in their ecological niche. Their biochemical complexity has profound implications for nutrition, immune system development, disease progression, and drug metabolism, as well as the variation in these processes that exists between individuals. While the species composition of the human microbiome has been deeply explored, detailed mechanistic studies linking specific microbial molecules to host phenotypes are still nascent. In this review, we discuss challenges in decoding these interaction networks, which require interdisciplinary approaches that combine chemical biology, microbiology, immunology, genetics, analytical chemistry, bioinformatics, and synthetic biology. We highlight important classes of microbiota-derived small molecules and notable examples. An understanding of these molecular mechanisms is central to realizing the potential of precision microbiome editing in health, disease, and therapeutic responses.
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http://dx.doi.org/10.1146/annurev-biochem-080320-115307DOI Listing
June 2021

Dual Targeting of v-ATPase and mTORC1 Signaling Disarms Multidrug-Resistant Cancers.

Cell Chem Biol 2020 11;27(11):1329-1331

Department of Chemistry, Yale University, New Haven, CT 06520, USA; Chemical Biology Institute, Yale University, West Haven, CT 06516, USA; Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06536, USA. Electronic address:

Chemotherapeutic treatments are frequently impeded by the development of multidrug resistance (MDR). In this issue of Cell Chemical Biology, Wang et al. (2020) identify the natural product verucopeptin as having therapeutic potential toward MDR cancer cell types by targeting v-ATPase and mTORC1 signaling.
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http://dx.doi.org/10.1016/j.chembiol.2020.10.013DOI Listing
November 2020

Phylogenetic and physiological signals in metazoan fossil biomolecules.

Sci Adv 2020 Jul 10;6(28):eaba6883. Epub 2020 Jul 10.

Department of Earth and Planetary Sciences, Yale University, New Haven, CT 06511, USA.

Proteins, lipids, and sugars establish animal form and function. However, the preservation of biological signals in fossil organic matter is poorly understood. Here, we used high-resolution in situ Raman microspectroscopy to analyze the molecular compositions of 113 Phanerozoic metazoan fossils and sediments. Proteins, lipids, and sugars converge in composition during fossilization through lipoxidation and glycoxidation to form endogenous N-, O-, and S-heterocyclic polymers. Nonetheless, multivariate spectral analysis reveals molecular heterogeneities: The relative abundance of glycoxidation and lipoxidation products distinguishes different tissue types. Preserved chelating ligands are diagnostic of different modes of biomineralization. Amino acid-specific fossilization products retain phylogenetic information and capture higher-rank metazoan relationships. Molecular signals survive in deep time and provide a powerful tool for reconstructing the evolutionary history of animals.
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http://dx.doi.org/10.1126/sciadv.aba6883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439315PMC
July 2020

Sulfamethoxazole drug stress upregulates antioxidant immunomodulatory metabolites in Escherichia coli.

Nat Microbiol 2020 11 27;5(11):1319-1329. Epub 2020 Jul 27.

Department of Chemistry, Yale University, New Haven, CT, USA.

Escherichia coli is an important model organism in microbiology and a prominent member of the human microbiota. Environmental isolates readily colonize the gastrointestinal tract of humans and other animals, and they can serve diverse probiotic, commensal and pathogenic roles in the host. Although certain strains have been associated with the severity of inflammatory bowel disease (IBD), the diverse immunomodulatory phenotypes remain largely unknown at the molecular level. Here, we decode a previously unknown E. coli metabolic pathway that produces a family of hybrid pterin-phenylpyruvate conjugates, which we named the colipterins. The metabolites are upregulated by subinhibitory levels of the antifolate sulfamethoxazole, which is used to treat infections including in patients with IBD. The genes folX/M and aspC/tyrB involved in monapterin biosynthesis and aromatic amino acid transamination, respectively, were required to initiate the colipterin pathway. We show that the colipterins are antioxidants, harbour diverse immunological activities in primary human tissues, activate anti-inflammatory interleukin-10 and improve colitis symptoms in a colitis mouse model. Our study defines an antifolate stress response in E. coli and links its associated metabolites to a major immunological marker of IBD.
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http://dx.doi.org/10.1038/s41564-020-0763-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581551PMC
November 2020

Making and Breaking Leupeptin Protease Inhibitors in Pathogenic Gammaproteobacteria.

Angew Chem Int Ed Engl 2020 10 13;59(41):17872-17880. Epub 2020 Aug 13.

Department of Chemistry, Yale University, New Haven, CT, 06520, USA.

Leupeptin is a bacterial small molecule that is used worldwide as a protease inhibitor. However, its biosynthesis and genetic distribution remain unknown. We identified a family of leupeptins in gammaproteobacterial pathogens, including Photorhabdus, Xenorhabdus, and Klebsiella species, amongst others. Through genetic, metabolomic, and heterologous expression analyses, we established their construction by discretely expressed ligases and accessory enzymes. In Photorhabdus species, a hypothetical protein required for colonizing nematode hosts was established as a new class of proteases. This enzyme cleaved the tripeptide aldehyde protease inhibitors, leading to the formation of "pro-pyrazinones" featuring a hetero-tricyclic architecture. In Klebsiella oxytoca, the pathway was enriched in clinical isolates associated with respiratory tract infections. Thus, the bacterial production and proteolytic degradation of leupeptins can be associated with animal colonization phenotypes.
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http://dx.doi.org/10.1002/anie.202005506DOI Listing
October 2020

Structure and bioactivity of colibactin.

Bioorg Med Chem Lett 2020 08 23;30(15):127280. Epub 2020 May 23.

Department of Chemistry, Yale University, New Haven, CT 06520, United States; Department of Pharmacology, Yale School of Medicine, New Haven, CT 06520, United States. Electronic address:

Colibactin is a secondary metabolite produced by certain strains of bacteria found in the human gut. The presence of colibactin-producing bacteria has been correlated to colorectal cancer in humans. Colibactin was first discovered in 2006, but because it is produced in small quantities and is unstable, it has yet to be isolated from bacterial cultures. Here we summarize advances in the field since ~2017 that have led to the identification of the structure of colibactin as a heterodimer containing two DNA-reactive electrophilic cyclopropane residues. Colibactin has been shown to form interstrand cross-links by alkylation of adenine residues on opposing strands of DNA. The structure of colibactin contains two thiazole rings separated by a two-carbon linker that is thought to exist as an α-aminoketone following completion of the biosynthetic pathway. However, synthetic studies have now established that this α-aminoketone is unstable toward aerobic oxidation; the resulting oxidation products are in turn unstable toward nucleophilic cleavage under mild conditions. These data provide a simple molecular-level explanation for colibactin's instability and potentially also explain the observation that cell-to-cell contact is required for genotoxic effects.
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http://dx.doi.org/10.1016/j.bmcl.2020.127280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309967PMC
August 2020

Dimeric Stilbene Antibiotics Target the Bacterial Cell Wall in Drug-Resistant Gram-Positive Pathogens.

Biochemistry 2020 06 20;59(21):1966-1971. Epub 2020 May 20.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

The prevalence of antibiotic resistance has been increasing globally, and new antimicrobial agents are needed to address this growing problem. We previously reported that a stilbene dimer from gammaproteobacteria exhibits strong activity relative to its monomer against the multidrug-resistant Gram-positive pathogens methicillin-resistant (MRSA) and vancomycin-resistant . Here, we show that related dietary plant stilbene-derived dimers also have activity against these pathogens, and MRSA is unable to develop substantial resistance even after daily nonlethal exposure to the lead compound for a duration of three months. Through a systematic deduction process, we established the mode of action of the lead dimer, which targets the bacterial cell wall. Genome sequencing of modest resistance mutants, mass spectrometry analysis of cell wall precursors, and exogenous lipid II chemical complementation studies support the target as being lipid II itself or lipid II trafficking processes. Given the broad distribution of stilbenes in plants, including dietary plants, we anticipate that our mode of action studies here could be more broadly applicable to multipartite host-bacterium-plant interactions.
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http://dx.doi.org/10.1021/acs.biochem.0c00213DOI Listing
June 2020

A DNA Repair Inhibitor Isolated from an Ecuadorian Fungal Endophyte Exhibits Synthetic Lethality in PTEN-Deficient Glioblastoma.

J Nat Prod 2020 06 14;83(6):1899-1908. Epub 2020 May 14.

Department of Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut 06520, United States.

Disruption of the tumor suppressor PTEN, either at the protein or genomic level, plays an important role in human cancer development. The high frequency of PTEN deficiency reported across several cancer subtypes positions therapeutic approaches that exploit PTEN loss-of-function with the ability to significantly impact the treatment strategies of a large patient population. Here, we report that an endophytic fungus isolated from a medicinal plant produces an inhibitor of DNA double-strand-break repair. Furthermore, the novel alkaloid product, which we have named irrepairzepine (), demonstrated synthetic lethal targeting in PTEN-deficient glioblastoma cells. Our results uncover a new therapeutic lead for PTEN-deficient cancers and an important molecular tool toward enhancing the efficacy of current cancer treatments.
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http://dx.doi.org/10.1021/acs.jnatprod.0c00012DOI Listing
June 2020

Cellular Stress Upregulates Indole Signaling Metabolites in Escherichia coli.

Cell Chem Biol 2020 06 2;27(6):698-707.e7. Epub 2020 Apr 2.

Department of Chemistry, Yale University, New Haven, CT 06520, USA; Chemical Biology Institute, Yale University, West Haven, CT 06516, USA; Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT 06536, USA. Electronic address:

Escherichia coli broadly colonize the intestinal tract of humans and produce a variety of small molecule signals. However, many of these small molecules remain unknown. Here, we describe a family of widely distributed bacterial metabolites termed the "indolokines." In E. coli, the indolokines are upregulated in response to a redox stressor via aspC and tyrB transaminases. Although indolokine 1 represents a previously unreported metabolite, four of the indolokines (2-5) were previously shown to be derived from indole-3-carbonyl nitrile (ICN) in the plant pathogen defense response. We show that the indolokines are produced in a convergent evolutionary manner relative to plants, enhance E. coli persister cell formation, outperform ICN protection in an Arabidopsis thaliana-Pseudomonas syringae infection model, trigger a hallmark plant innate immune response, and activate distinct immunological responses in primary human tissues. Our molecular studies link a family of cellular stress-induced metabolites to defensive responses across bacteria, plants, and humans.
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http://dx.doi.org/10.1016/j.chembiol.2020.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306003PMC
June 2020

Characterization of Autoinducer-3 Structure and Biosynthesis in .

ACS Cent Sci 2020 Feb 22;6(2):197-206. Epub 2020 Jan 22.

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.

is a common inhabitant of the human microbiota and a beacon model organism in biology. However, an understanding of its signaling systems that regulate population-level phenotypes known as quorum sensing remain incomplete. Here, we define the structure and biosynthesis of autoinducer-3 (AI-3), a metabolite of previously unknown structure involved in the pathogenesis of enterohemorrhagic (EHEC). We demonstrate that novel AI-3 analogs are derived from threonine dehydrogenase (Tdh) products and "abortive" tRNA synthetase reactions, and they are distributed across a variety of Gram-negative and Gram-positive bacterial pathogens. In addition to regulating virulence genes in EHEC, we show that the metabolites exert diverse immunological effects on primary human tissues. The discovery of AI-3 metabolites and their biochemical origins now provides a molecular foundation for investigating the diverse biological roles of these elusive yet widely distributed bacterial signaling molecules.
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http://dx.doi.org/10.1021/acscentsci.9b01076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047286PMC
February 2020

Bacterial Autoimmune Drug Metabolism Transforms an Immunomodulator into Structurally and Functionally Divergent Antibiotics.

Angew Chem Int Ed Engl 2020 05 17;59(20):7871-7880. Epub 2020 Mar 17.

Department of Chemistry, Yale University, New Haven, CT, 06520, USA.

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.
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http://dx.doi.org/10.1002/anie.201916204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200298PMC
May 2020

Addendum: Synthesis and reactivity of precolibactin 886.

Nat Chem 2019 12;11(12):1167

Department of Chemistry, Yale University, New Haven, CT, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41557-019-0383-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523535PMC
December 2019

Synthesis and reactivity of precolibactin 886.

Nat Chem 2019 10 23;11(10):890-898. Epub 2019 Sep 23.

Department of Chemistry, Yale University, New Haven, CT, USA.

The clb gene cluster encodes the biosynthesis of metabolites known as precolibactins and colibactins. The clb pathway is found in gut commensal Escherichia coli, and clb metabolites are thought to initiate colorectal cancer via DNA crosslinking. Here we report confirmation of the structural assignment of the complex clb product precolibactin 886 via a biomimetic synthetic pathway. We show that an α-ketoimine linear precursor undergoes spontaneous cyclization to precolibactin 886 on HPLC purification. Studies of this α-ketoimine and the related α-dicarbonyl revealed that these compounds are unexpectedly susceptible to nucleophilic cleavage under mildly basic conditions. This cleavage pathway forms other known clb metabolites or biosynthetic intermediates and explains the difficulties in isolating fully mature biosynthetic products. This cleavage also accounts for a recently identified colibactin-adenine adduct. The colibactin peptidase ClbP deacylates synthetic precolibactin 886 to form a non-genotoxic pyridone, which suggests precolibactin 886 lies off the path of the major biosynthetic route.
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http://dx.doi.org/10.1038/s41557-019-0338-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761996PMC
October 2019

An Ugi-like Biosynthetic Pathway Encodes Bombesin Receptor Subtype-3 Agonists.

J Am Chem Soc 2019 10 2;141(41):16271-16278. Epub 2019 Oct 2.

Department of Chemistry , Yale University , New Haven , Connecticut 06520 , United States.

Isocyanide functional groups can be found in a variety of natural products. Rhabduscin is one such isocyanide-functionalized immunosuppressant produced in and gammaproteobacterial pathogens, and deletion of its biosynthetic gene cluster inhibits virulence in an invertebrate animal infection model. Here, we characterized the first "opine-glycopeptide" class of natural products termed rhabdoplanins, and strikingly, these molecules are spontaneously produced from rhabduscin via an unprecedented multicomponent "Ugi-like" reaction sequence in nature. The rhabdoplanins also represent new lead G protein-coupled receptor (GPCR) agonists, stimulating the bombesin receptor subtype-3 (BB3) GPCR.
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http://dx.doi.org/10.1021/jacs.9b04183DOI Listing
October 2019

Bright Green Biofluorescence in Sharks Derives from Bromo-Kynurenine Metabolism.

iScience 2019 Sep 8;19:1291-1336. Epub 2019 Aug 8.

Department of Chemistry, Yale University, New Haven, CT 06520, USA; Chemical Biology Institute, Yale University, West Haven, CT 06516, USA; Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT 06536, USA. Electronic address:

Although in recent years there has been an increased awareness of the widespread nature of biofluorescence in the marine environment, the diversity of the molecules responsible for this luminescent phenotype has been mostly limited to green fluorescent proteins (GFPs), GFP-like proteins, and fluorescent fatty acid-binding proteins (FABPs). In the present study, we describe a previously undescribed group of brominated tryptophan-kynurenine small molecule metabolites responsible for the green biofluorescence in two species of sharks and provide their structural, antimicrobial, and spectral characterization. Multi-scale fluorescence microscopy studies guided the discovery of metabolites that were differentially produced in fluorescent versus non-fluorescent skin, as well as the species-specific structural details of their unusual light-guiding denticles. Overall, this study provides the detailed description of a family of small molecules responsible for marine biofluorescence and opens new questions related to their roles in central nervous system signaling, resilience to microbial infections, and photoprotection.
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http://dx.doi.org/10.1016/j.isci.2019.07.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831821PMC
September 2019

Structure elucidation of colibactin and its DNA cross-links.

Science 2019 09 8;365(6457). Epub 2019 Aug 8.

Department of Chemistry, Yale University, New Haven, CT 06520, USA.

Colibactin is a complex secondary metabolite produced by some genotoxic gut strains. The presence of colibactin-producing bacteria correlates with the frequency and severity of colorectal cancer in humans. However, because colibactin has not been isolated or structurally characterized, studying the physiological effects of colibactin-producing bacteria in the human gut has been difficult. We used a combination of genetics, isotope labeling, tandem mass spectrometry, and chemical synthesis to deduce the structure of colibactin. Our structural assignment accounts for all known biosynthetic and cell biology data and suggests roles for the final unaccounted enzymes in the colibactin gene cluster.
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http://dx.doi.org/10.1126/science.aax2685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6820679PMC
September 2019

Biocatalytic Reversal of Advanced Glycation End Product Modification.

Chembiochem 2019 09 9;20(18):2402-2410. Epub 2019 Aug 9.

Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT, 06511, USA.

Advanced glycation end products (AGEs) are a heterogeneous group of molecules that emerge from the condensation of sugars and proteins through the Maillard reaction. Despite a significant number of studies showing strong associations between AGEs and the pathologies of aging-related illnesses, it has been a challenge to establish AGEs as causal agents primarily due to the lack of tools in reversing AGE modifications at the molecular level. Herein, we show that MnmC, an enzyme involved in a bacterial tRNA-modification pathway, is capable of reversing the AGEs carboxyethyl-lysine (CEL) and carboxymethyl-lysine (CML) back to their native lysine structure. Combining structural homology analysis, site-directed mutagenesis, and protein domain dissection studies, we generated a variant of MnmC with improved catalytic properties against CEL in its free amino acid form. We show that this enzyme variant is also active on a CEL-modified peptidomimetic and an AGE-containing peptide that has been established as an authentic ligand of the receptor for AGEs (RAGE). Our data demonstrate that MnmC variants are promising lead catalysts toward the development of AGE-reversal tools and a better understanding of AGE biology.
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http://dx.doi.org/10.1002/cbic.201900158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6768434PMC
September 2019

A Forward Chemical Genetic Screen Reveals Gut Microbiota Metabolites That Modulate Host Physiology.

Cell 2019 05 18;177(5):1217-1231.e18. Epub 2019 Apr 18.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA. Electronic address:

The intestinal microbiota produces tens of thousands of metabolites. Here, we used host sensing of small molecules by G-protein coupled receptors (GPCRs) as a lens to illuminate bioactive microbial metabolites that impact host physiology. We screened 144 human gut bacteria against the non-olfactory GPCRome and identified dozens of bacteria that activated both well-characterized and orphan GPCRs, including strains that converted dietary histidine into histamine and shaped colonic motility; a prolific producer of the essential amino acid L-Phe, which we identified as an agonist for GPR56 and GPR97; and a species that converted L-Phe into the potent psychoactive trace amine phenethylamine, which crosses the blood-brain barrier and triggers lethal phenethylamine poisoning after monoamine oxidase inhibitor administration. These studies establish an orthogonal approach for parsing the microbiota metabolome and uncover multiple biologically relevant host-microbiota metabolome interactions.
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http://dx.doi.org/10.1016/j.cell.2019.03.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536006PMC
May 2019

The Gut Microbiome Says NO to microRNA-Mediated Gene Silencing.

Biochemistry 2019 04 5;58(16):2089-2090. Epub 2019 Apr 5.

Chemical Biology Institute , Yale University , West Haven , Connecticut 06516 , United States.

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http://dx.doi.org/10.1021/acs.biochem.9b00262DOI Listing
April 2019

Bacterial Analogs of Plant Tetrahydropyridine Alkaloids Mediate Microbial Interactions in a Rhizosphere Model System.

Appl Environ Microbiol 2019 05 2;85(10). Epub 2019 May 2.

Wisconsin Institute for Discovery and Department of Plant Pathology, University of Wisconsin-Madison, Madison, Wisconsin, USA

Plants expend significant resources to select and maintain rhizosphere communities that benefit their growth and protect them from pathogens. A better understanding of assembly and function of rhizosphere microbial communities will provide new avenues for improving crop production. Secretion of antibiotics is one means by which bacteria interact with neighboring microbes and sometimes change community composition. In our analysis of a taxonomically diverse consortium from the soybean rhizosphere, we found that selectively inhibits growth of and other members of the grown in soybean root exudate. A genetic screen in identified a previously uncharacterized biosynthetic gene cluster responsible for the inhibitory activity. Metabolites were isolated based on biological activity and were characterized using tandem mass spectrometry, multidimensional nuclear magnetic resonance, and Mosher ester analysis, leading to the discovery of a new family of bacterial tetrahydropyridine alkaloids, koreenceine A to D (metabolites 1 to 4). Three of these metabolites are analogs of the plant alkaloid γ-coniceine. Comparative analysis of the koreenceine cluster with the γ-coniceine pathway revealed distinct polyketide synthase routes to the defining tetrahydropyridine scaffold, suggesting convergent evolution. Koreenceine-type pathways are widely distributed among species, and koreenceine C was detected in another species from a distantly related cluster. This work suggests that and plants convergently evolved the ability to produce similar alkaloid metabolites that can mediate interbacterial competition in the rhizosphere. The microbiomes of plants are critical to host physiology and development. Microbes are attracted to the rhizosphere due to massive secretion of plant photosynthates from roots. Microorganisms that successfully join the rhizosphere community from bulk soil have access to more abundant and diverse molecules, producing a highly competitive and selective environment. In the rhizosphere, as in other microbiomes, little is known about the genetic basis for individual species' behaviors within the community. In this study, we characterized competition between and , two common rhizosphere inhabitants. We identified a widespread gene cluster in several spp. that is necessary for the production of a novel family of tetrahydropyridine alkaloids that are structural analogs of plant alkaloids. We expand the known repertoire of antibiotics produced by in the rhizosphere and demonstrate the role of the metabolites in interactions with other rhizosphere bacteria.
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http://dx.doi.org/10.1128/AEM.03058-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498172PMC
May 2019

Introducing THOR, a Model Microbiome for Genetic Dissection of Community Behavior.

mBio 2019 03 5;10(2). Epub 2019 Mar 5.

Wisconsin Institute for Discovery and Department of Plant Pathology, University of Wisconsin-Madison, Madison, Wisconsin, USA

The quest to manipulate microbiomes has intensified, but many microbial communities have proven to be recalcitrant to sustained change. Developing model communities amenable to genetic dissection will underpin successful strategies for shaping microbiomes by advancing an understanding of community interactions. We developed a model community with representatives from three dominant rhizosphere taxa, the , , and We chose as a model rhizosphere firmicute and characterized 20 other candidates, including "hitchhikers" that coisolated with from the rhizosphere. Pairwise analysis produced a hierarchical interstrain-competition network. We chose two hitchhikers, from the top tier of the competition network and from the bottom of the network, to represent the and , respectively. The model community has several emergent properties, induction of dendritic expansion of colonies by either of the other members, and production of more robust biofilms by the three members together than individually. Moreover, produces a novel family of alkaloid antibiotics that inhibit growth of , and production is inhibited by We designate this community THOR, because the members are he itchhikers f the hizosphere. The genetic, genomic, and biochemical tools available for dissection of THOR provide the means to achieve a new level of understanding of microbial community behavior. The manipulation and engineering of microbiomes could lead to improved human health, environmental sustainability, and agricultural productivity. However, microbiomes have proven difficult to alter in predictable ways, and their emergent properties are poorly understood. The history of biology has demonstrated the power of model systems to understand complex problems such as gene expression or development. Therefore, a defined and genetically tractable model community would be useful to dissect microbiome assembly, maintenance, and processes. We have developed a tractable model rhizosphere microbiome, designated THOR, containing , , and , which represent three dominant phyla in the rhizosphere, as well as in soil and the mammalian gut. The model community demonstrates emergent properties, and the members are amenable to genetic dissection. We propose that THOR will be a useful model for investigations of community-level interactions.
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http://dx.doi.org/10.1128/mBio.02846-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401489PMC
March 2019

Characterization of a Hybrid Nonribosomal Peptide-Carbohydrate Biosynthetic Pathway in Photorhabdus luminescens.

Biochemistry 2019 02 13;58(8):1131-1140. Epub 2019 Feb 13.

Department of Chemistry , Yale University , New Haven , Connecticut 06520 , United States.

Advances in genome sequencing and analysis have afforded a trove of "orphan" bacterial biosynthetic pathways, many of which contain hypothetical proteins. Given the potential for these hypothetical proteins to carry out novel chemistry, orphan pathways serve as a rich reservoir for the discovery of new enzymes responsible for the production of metabolites with both fascinating chemistries and biological functions. We previously identified a rare hybrid nonribosomal peptide synthetase (NRPS)-carbohydrate genomic island in the entomopathogen Photorhabdus luminescens. Heterologous expression of the pathway led to the characterization of oligosaccharides harboring a 1,6-anhydro-β-d- N-acetyl-glucosamine moiety, but these new metabolites lacked modification by the NRPS machinery. Here, through the application of top-down protein mass spectrometry, pathway-targeted molecular networking, stable isotope labeling, and in vitro protein biochemistry, we complete the characterization of this biosynthetic pathway and identify the hybrid product of the pathway, a new "glycoamino acid" metabolite termed photolose. Intriguingly, a hypothetical protein served as a bridge to condense a glycyl unit derived from the NRPS machinery onto the free 1,6-anhydro-β-d- N-acetyl-glucosamine core. We further demonstrate that the gene cluster confers a growth advantage to antimicrobial peptide challenge.
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http://dx.doi.org/10.1021/acs.biochem.8b01120DOI Listing
February 2019

Model Colibactins Exhibit Human Cell Genotoxicity in the Absence of Host Bacteria.

ACS Chem Biol 2018 12 20;13(12):3286-3293. Epub 2018 Nov 20.

Department of Microbial Pathogenesis , Yale School of Medicine , New Haven , Connecticut 06536 , United States.

Colibactins are genotoxic secondary metabolites produced in select Enterobacteriaceae, which induce downstream DNA double-strand breaks (DSBs) in human cell lines and are thought to promote the formation of colorectal tumors. Although key structural and functional features of colibactins have been elucidated, the full molecular mechanisms regulating these phenotypes remain unknown. Here, we demonstrate that free model colibactins induce DSBs in human cell cultures and do not require delivery by host bacteria. Through domain-targeted editing, we demonstrate that a subset of native colibactins generated from observed module skipping in the nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) biosynthetic assembly line share DNA alkylation phenotypes with the model colibactins in vitro. However, module skipping eliminates the strong DNA interstrand cross-links formed by the wild-type pathway in cell culture. This product diversification during the modular NRPS-PKS biosynthesis produces a family of metabolites with varying observed mechanisms of action (DNA alkylation versus cross-linking) in cell culture. The presence of membranes separating human cells from model colibactins attenuated genotoxicity, suggesting that membrane diffusion limits colibactin activity and could account for the reported bacterium-human cell-to-cell contact phenotype. Additionally, extracellular supplementation of the colibactin resistance protein ClbS was able to intercept colibactins in an Escherichia coli-human cell transient infection model. Our studies demonstrate that free model colibactins recapitulate cellular phenotypes associated with module-skipped products in the native colibactin pathway and define specific protein domains that are required for efficient DNA interstrand cross-linking in the native pathway.
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http://dx.doi.org/10.1021/acschembio.8b00714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001666PMC
December 2018

Characterization of Natural Colibactin-Nucleobase Adducts by Tandem Mass Spectrometry and Isotopic Labeling. Support for DNA Alkylation by Cyclopropane Ring Opening.

Biochemistry 2018 11 31;57(45):6391-6394. Epub 2018 Oct 31.

Department of Chemistry , Yale University , New Haven , Connecticut 06520 , United States.

Colibactins are genotoxic secondary metabolites whose biosynthesis is encoded in the clb gene cluster harbored by certain strains of gut commensal Escherichia coli. Using synthetic colibactin analogues, we previously provided evidence that colibactins alkylate DNA by addition of a nucleotide to an electrophilic cyclopropane intermediate. However, natural colibactin-nucleobase adducts have not been identified, to the best of our knowledge. Here we present the first identification of such adducts, derived from treatment of pUC19 DNA with clb E. coli. Previous biosynthetic studies established cysteine and methionine as building blocks in colibactin biosynthesis; accordingly, we used cysteine (Δ cysE) and methionine (Δ metA) auxotrophic strains cultured in media supplemented with l-[U-C]Cys or l-[U-C]Met to facilitate the identification of nucleobases bound to colibactins. Using MS and MS analysis, in conjunction with the known oxidative instability of colibactin cyclopropane-opened products, we were able to characterize adenine adducts derived from cyclopropane ring opening. This study provides the first reported detection of nucleobase adducts derived from clb E. coli and lends support to our earlier model suggesting DNA alkylation by addition of a nucleotide to an electrophilic cyclopropane.
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http://dx.doi.org/10.1021/acs.biochem.8b01023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997931PMC
November 2018

Luciferin production and luciferase transcription in the bioluminescent copepod .

PeerJ 2018 14;6:e5506. Epub 2018 Sep 14.

Department of Natural Sciences, City University of New York, Bernard M. Baruch College, New York, NY, United States of America.

Bioluminescent copepods are often the most abundant marine zooplankton and play critical roles in oceanic food webs. copepods exhibit particularly bright bioluminescence, and the molecular basis of their light production has just recently begun to be explored. Here we add to this body of work by transcriptomically profiling , a common species found in temperate, northern, and southern latitudes. In this previously molecularly-uncharacterized species, we find the typical luciferase paralog gene set found in . More surprisingly, we recover noteworthy putative luciferase sequences that had not been described from species, indicating that bioluminescence produced by these copepods may be more complex than previously known. This includes another copepod luciferase, as well as one from a shrimp. Furthermore, feeding experiments using mass spectrometry and C labelled L-tyrosine and L-phenylalanine firmly establish that produces its own coelenterazine luciferin rather than acquiring it through diet. This coelenterazine synthesis has only been directly confirmed in one other copepod species.
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http://dx.doi.org/10.7717/peerj.5506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140675PMC
September 2018
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