Jon Clardy

Harvard Medical School, Boston, Massachusetts, United States

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Publications (604)1852.58 Total impact

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    ABSTRACT: Roseobacticides regulate the symbiotic relationship between a marine bacterium (Phaeobacter inhibens) and a marine microalga (Emiliania huxleyi). This relationship can be mutualistic, when the algal host provides food for the bacteria and the bacteria produce growth hormones and antibiotics for the algae, or parasitic, when the algae senesce and release p-coumaric acid. The released p-coumaric acid causes the bacteria to synthesize roseobacticides, which are nM-μM toxins for the algae. We examined the biosynthesis of roseobacticides and report that all roseobacticide precursors play critical roles during the mutualist phase of the symbiosis. Roseobacticides are biosynthesized from the algal growth promoter, the major food molecule provided by the algal cells, and the algal senescence signal that initiates the mutualist-to-parasite switch. Thus, molecules that are beneficial during mutualism are diverted to the synthesis of toxins during parasitism. A plausible mechanism for assembling roseobacticides from these molecules is proposed.
    Journal of the American Chemical Society 10/2014; · 10.68 Impact Factor
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    ABSTRACT: In complex biological systems, small molecules often mediate microbe-microbe and microbe-host interactions. Using a systematic approach, we identified 3,118 small-molecule biosynthetic gene clusters (BGCs) in genomes of human-associated bacteria and studied their representation in 752 metagenomic samples from the NIH Human Microbiome Project. Remarkably, we discovered that BGCs for a class of antibiotics in clinical trials, thiopeptides, are widely distributed in genomes and metagenomes of the human microbiota. We purified and solved the structure of a thiopeptide antibiotic, lactocillin, from a prominent member of the vaginal microbiota. We demonstrate that lactocillin has potent antibacterial activity against a range of Gram-positive vaginal pathogens, and we show that lactocillin and other thiopeptide BGCs are expressed in vivo by analyzing human metatranscriptomic sequencing data. Our findings illustrate the widespread distribution of small-molecule-encoding BGCs in the human microbiome, and they demonstrate the bacterial production of drug-like molecules in humans. PAPERCLIP:
    Cell 09/2014; 158(6):1402-14. · 31.96 Impact Factor
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    ABSTRACT: This report describes complete practical guidelines and insights for the crystalline sponge method, which have been derived through the first use of synchrotron radiation on these systems, and includes a procedure for faster synthesis of the sponges. These guidelines will be applicable to crystal sponge data collected at synchrotrons or in-house facilities, and will allow researchers to obtain reliable high-quality data and construct chemically and physically sensible models for guest structural determination.
    Acta Crystallographica Section A: Foundations and Advances. 08/2014;
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    ABSTRACT: Malaria, an infectious disease caused by eukaryotic parasites of the genus Plasmodium, afflicts hundreds of millions of people every year. Both the parasite and its host utilize protein kinases to regulate essential cellular processes. Bioinformatic analyses of parasite genomes predict at least 65 protein kinases, but their biological functions and therapeutic potential are largely unknown. We profiled 1358 small-molecule kinase inhibitors to evaluate the role of both the human and the malaria kinomes in Plasmodium infection of liver cells, the parasites' obligatory but transient developmental stage that precedes the symptomatic blood stage. The screen identified several small molecules that inhibit parasite load in liver cells, some with nanomolar efficacy, and each compound was subsequently assessed for activity against blood-stage malaria. Most of the screening hits inhibited both liver- and blood-stage malaria parasites, which have dissimilar gene expression profiles and infect different host cells. Evaluation of existing kinase activity profiling data for the library members suggests that several kinases are essential to malaria parasites, including cyclin-dependent kinases (CDKs), glycogen synthase kinases, and phosphoinositide-3-kinases. CDK inhibitors were found to bind to Plasmodium protein kinase 5, but it is likely that these compounds target multiple parasite kinases. The dual-stage inhibition of the identified kinase inhibitors makes them useful chemical probes and promising starting points for antimalarial development.
    ChemBioChem 08/2014; · 3.74 Impact Factor
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    ABSTRACT: Biochemical high-throughput screening is widely used in drug discovery, using a variety of small molecule libraries. However, broader screening strategies may be more beneficial to identify novel biological mechanisms. In the current study we utilized a β-galactosidase complementation method to screen a selection of microbial-derived pre-fractionated natural product extracts for those that increase Regulator of G protein Signaling 2 (RGS2) protein levels. RGS2 is a member of a large family of proteins that all regulate signaling through G protein-coupled receptors (GPCRs) by accelerating GTPase activity on active Gα as well as through other mechanisms. RGS2(-/-) mice are hypertensive, show increased anxiety and are prone to heart failure. RGS2 has a very short protein half-life due to rapid proteasomal degradation and we propose that enhancement of RGS2 protein levels could be a beneficial therapeutic strategy. Bioassay-guided fractionation of one of the hit strains yielded a pure compound, Indolactam V, a known protein kinase C (PKC) activator, which selectively increased RGS2 protein levels in a time- and concentration-dependent manner. Similar results were obtained with phorbol 12-myristate 13-acetate (PMA) as well as activation of the Gq-coupled muscarinic M3 receptor. The effect on RGS2 protein levels was blocked by the non-selective PKC inhibitor Go6983, the PKCβ-selective inhibitor Ruboxastaurin, as well as siRNA-mediated knockdown of PKCβ. Indolactam V-mediated increases in RGS2 protein levels also had functional effects on GPCR signaling. This study provides important proof-of-concept for our screening strategy and could define a negative feedback mechanism in Gq/PLC signaling through RGS2 protein up-regulation.
    Molecular pharmacology. 08/2014;
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    ABSTRACT: Although biosynthetic gene clusters (BGCs) have been discovered for hundreds of bacterial metabolites, our knowledge of their diversity remains limited. Here, we used a novel algorithm to systematically identify BGCs in the extensive extant microbial sequencing data. Network analysis of the predicted BGCs revealed large gene cluster families, the vast majority uncharacterized. We experimentally characterized the most prominent family, consisting of two subfamilies of hundreds of BGCs distributed throughout the Proteobacteria; their products are aryl polyenes, lipids with an aryl head group conjugated to a polyene tail. We identified a distant relationship to a third subfamily of aryl polyene BGCs, and together the three subfamilies represent the largest known family of biosynthetic gene clusters, with more than 1,000 members. Although these clusters are widely divergent in sequence, their small molecule products are remarkably conserved, indicating for the first time the important roles these compounds play in Gram-negative cell biology.
    Cell. 07/2014; 158(2):412-21.
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    ABSTRACT: Studies on the origin of animal multicellularity have increasingly focused on one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta. Single cells of S. rosetta can develop into multicellular rosette-shaped colonies through a process of incomplete cytokinesis. Unexpectedly, the initiation of rosette development requires bacterially produced small molecules. Previously, our laboratories reported the planar structure and femtomolar rosette-inducing activity of one rosette-inducing small molecule, dubbed rosette-inducing factor 1 (RIF-1), produced by the Gram-negative Bacteroidetes bacterium Algoriphagus machipongonensis. RIF-1 belongs to the small and poorly explored class of sulfonolipids. Here, we report a modular total synthesis of RIF-1 stereoisomers and structural analogs. Rosette-induction assays using synthetic RIF 1 stereoisomers and naturally occurring analogs defined the absolute stereochemistry of RIF-1 and revealed a remarkably restrictive set of structural requirements for inducing rosette development.
    Journal of the American Chemical Society 07/2014; · 10.68 Impact Factor
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    ABSTRACT: Actinobacteria in the genus Streptomyces are critical players in microbial communities that decompose complex carbohydrates in the soil, and these bacteria have recently been implicated in the deconstruction of plant polysaccharides for some herbivorous insects. Despite the importance of Streptomyces to carbon cycling, the extent of their plant biomass degrading ability remain largely unknown. Here, we compared four strains of Streptomyces isolated from insect herbivores that attack pine trees: S. sp. DpondAA-B6 (SDPB6) from the mountain pine beetle; S. sp. SPB74 from the southern pine beetle; and S. sp. SirexAA-E (SACTE) and S. sp. SirexAA-G from the woodwasp, Sirex noctilio. Biochemical analysis of secreted enzymes demonstrated that only two of these strains, SACTE and SDPB6, were efficient at degrading plant biomass. Genomic analyses indicated that SACTE and SDPB6 are closely phylogenetically related and that they share similar compositions of carbohydrate active enzymes. Genome-wide proteomic and transcriptomic analyses revealed that the major exocellulases (GH6 and GH48), lytic polysaccharide monooxygenases (AA10), and mannanases (GH5) were conserved and secreted in both organisms, while the secreted endocellulases (GH5 and GH9 versus GH9 and GH12) were from diverged enzyme families. Together, these data identify two phylogenetically related insect-associated Streptomyces strains with high biomass degrading activity and characterize key enzymatic similarities and differences used by these organisms to deconstruct plant biomass.
    Applied and environmental microbiology. 05/2014;
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    Thomas Böttcher, Jon Clardy
    Angewandte Chemie 05/2014; 126(20).
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    ABSTRACT: Languishing antibiotic discovery and flourishing antibiotic resistance have prompted the development of alternative untapped sources for antibiotic discovery, including previously uncultured bacteria. Here, we screen extracts from uncultured species against Mycobacterium tuberculosis and identify lassomycin, an antibiotic that exhibits potent bactericidal activity against both growing and dormant mycobacteria, including drug-resistant forms of M. tuberculosis, but little activity against other bacteria or mammalian cells. Lassomycin is a highly basic, ribosomally encoded cyclic peptide with an unusual structural fold that only partially resembles that of other lasso peptides. We show that lassomycin binds to a highly acidic region of the ClpC1 ATPase complex and markedly stimulates its ATPase activity without stimulating ClpP1P2-catalyzed protein breakdown, which is essential for viability of mycobacteria. This mechanism, uncoupling ATPase from proteolytic activity, accounts for the bactericidal activity of lassomycin.
    Chemistry & biology 03/2014; · 6.52 Impact Factor
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    Thomas Böttcher, Jon Clardy
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    ABSTRACT: Some bacteria swarm under some circumstances; they move rapidly and collectively over a surface. In an effort to understand the molecular signals controlling swarming, we isolated two bacterial strains from the same red seaweed, Vibrio alginolyticus B522, a vigorous swarmer, and Shewanella algae B516, which inhibits V. alginolyticus swarming in its vicinity. Plate assays combined with NMR, MS, and X-ray diffraction analyses identified a small molecule, which was named avaroferrin, as a potent swarming inhibitor. Avaroferrin, a previously unreported cyclic dihydroxamate siderophore, is a chimera of two well-known siderophores: putrebactin and bisucaberin. The sequenced genome of S. algae revealed avaroferrin's biosynthetic gene cluster to be a mashup of putrebactin and bisucaberin biosynthetic genes. Avaroferrin blocks swarming through its ability to bind iron in a form that cannot be pirated by V. alginolyticus, thereby securing this essential resource for its producer.
    Angewandte Chemie International Edition 02/2014; · 11.34 Impact Factor
  • Pierre Stallforth, Jon Clardy
    Proceedings of the National Academy of Sciences 02/2014; · 9.81 Impact Factor
  • ACS Synthetic Biology 01/2014; · 3.95 Impact Factor
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    ABSTRACT: We report a preliminary functional and complete structural characterization of a highly unusual geldanamycin analog, natalamycin A, that was isolated from Streptomyces strain M56 recovered from a South African nest of Macrotermes natalensis termites. Bioassay-guided fractionation based on antifungal activity led to the isolation of natalamycin A, and a combination of NMR spectroscopy and X-ray crystallographic analysis, including highly-accurate quantum-chemical NMR calculations on the largest and most conformationally-flexible system to date, revealed natalamycin A's three-dimensional solid- and solution-state structure. This structure along with the structures of related compounds isolated from the same source suggest a geldanamycin-like biosynthetic pathway with unusual post-PKS modifications.
    Chemical Science 01/2014; · 8.31 Impact Factor
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    ABSTRACT: Drugs that target both malaria's liver and blood stages will be needed to reduce the disease's substantial worldwide morbidity and mortality. Evaluation of a 259-member library of compounds that block proliferation of the blood stage of malaria revealed several scaffolds - dihydroquinazolinones, phenyldiazenylpyridines, piperazinyl methyl quinolones, and bis-benzimidazoles - with promising activity against the liver stage. Focused structure-activity studies on the dihydroquinazolinone scaffold revealed several molecules with excellent potency against both blood and liver stages. One promising early lead with dual activity is 2-(p-bromophenyl)-3-(2-(diethylamino)ethyl)-2,3-dihydroquinazolin-4(1H)-one with EC50 values of 0.46 μM and 0.34 μM against liver stage P. berghei ANKA and blood stage P. falciparum 3D7 parasites, respectively. Structure-activity relationships revealed that liver stage activity for this compound class requires a 3-dialkyl amino ethyl group and is abolished by substitution at the ortho-position of the phenyl moiety. These compounds have minimal toxicity to mammalian cells and are thus attractive compounds for further development.
    Antimicrobial Agents and Chemotherapy 12/2013; · 4.57 Impact Factor
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    ABSTRACT: As increased antibiotic resistance erodes the efficacy of currently used drugs, the need for new candidates with therapeutic potential grows. Although the majority of antibiotics in clinical use originated from natural products, mostly from environmental actinomycetes, high rediscovery rates, among other factors, have diminished the enthusiasm for continued exploration of this historically important source. Several well-studied insect agricultural systems have bacterial symbionts that have evolved to produce small molecules that suppress environmental pathogens. These molecules represent an underexplored reservoir of potentially useful antibiotics. This report describes the multilateral symbioses common to insect agricultural systems, the general strategy used for antibiotic discovery and pertinent examples from three farming systems: fungus-farming ants, southern pine beetles (SPBs) and fungus-growing termites.The Journal of Antibiotics advance online publication, 7 August 2013; doi:10.1038/ja.2013.77.
    The Journal of Antibiotics 08/2013; · 2.19 Impact Factor
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    ABSTRACT: Stable multipartite mutualistic associations require that all partners benefit. We show that a single mutational step is sufficient to turn a symbiotic bacterium from an inedible but host-beneficial secondary metabolite producer into a host food source. The bacteria's host is a "farmer" clone of the social amoeba Dictyostelium discoideum that carries and disperses bacteria during its spore stage. Associated with the farmer are two strains of Pseudomonas fluorescens, only one of which serves as a food source. The other strain produces diffusible small molecules: pyrrolnitrin, a known antifungal agent, and a chromene that potently enhances the farmer's spore production and depresses a nonfarmer's spore production. Genome sequence and phylogenetic analyses identify a derived point mutation in the food strain that generates a premature stop codon in a global activator (gacA), encoding the response regulator of a two-component regulatory system. Generation of a knockout mutant of this regulatory gene in the nonfood bacterial strain altered its secondary metabolite profile to match that of the food strain, and also, independently, converted it into a food source. These results suggest that a single mutation in an inedible ancestral strain that served a protective role converted it to a "domesticated" food source.
    Proceedings of the National Academy of Sciences 07/2013; · 9.81 Impact Factor
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    ABSTRACT: While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCerBf), which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (iNKT) cells. We demonstrate that α-GalCerBf has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis.
    PLoS Biology 07/2013; 11(7):e1001610. · 12.69 Impact Factor
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    ABSTRACT: Pharbinilic acid (1), the first naturally occurring allogibberic acid, was isolated from ethanol extracts of morning glory (Pharbitis nil) seeds. Its absolute configuration was determined by NOESY NMR and ECD experiments. Compound 1 showed weak cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cells and weakly inhibited nitric oxide production in lipopolysaccharide-activated BV-2 microglia cells.
    Journal of Natural Products 07/2013; · 3.29 Impact Factor
  • Pierre Stallforth, Jon Clardy
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    ABSTRACT: Crystalline 'sponges' offer a way to impose order on small molecules so that their structures can be solved by X-ray crystallography. This enables nanogram quantities of material to be analysed using the technique. See Article p.461
    Nature 03/2013; 495(7442):456-7. · 38.60 Impact Factor

Publication Stats

8k Citations
1,852.58 Total Impact Points

Institutions

  • 2004–2014
    • Harvard Medical School
      • Department of Biological Chemistry and Molecular Pharmacology
      Boston, Massachusetts, United States
    • Montana Tech of the University of Montana
      Butte, Montana, United States
  • 2009–2012
    • Yale University
      • Department of Chemistry
      New Haven, CT, United States
    • Massachusetts General Hospital
      • Department of Molecular Biology
      Boston, MA, United States
  • 1981–2012
    • Harvard University
      • • Department of Molecular and Cell Biology
      • • Department of Immunology and Infectious Diseases
      • • Department of Chemistry and Chemical Biology
      Cambridge, MA, United States
  • 2008–2011
    • University of Wisconsin, Madison
      • Department of Bacteriology
      Madison, MS, United States
  • 2010
    • University of British Columbia - Vancouver
      Vancouver, British Columbia, Canada
    • Cheikh Anta Diop University, Dakar
      Dakar, Dakar, Senegal
  • 1979–2010
    • Cornell University
      • Department of Chemistry and Chemical Biology
      Ithaca, New York, United States
  • 2008–2009
    • Broad Institute of MIT and Harvard
      Cambridge, Massachusetts, United States
  • 2006–2008
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2007
    • The Rockefeller University
      • Laboratory of Genetically Encoded Small Molecules
      New York City, NY, United States
  • 1994–2006
    • University of California, Santa Cruz
      • Department of Chemistry & Biochemistry
      Santa Cruz, CA, United States
  • 1974–2004
    • Iowa State University
      • Department of Chemistry
      Ames, IA, United States
  • 2003
    • University of California, San Diego
      • Center for Marine Biotechnology and Biomedicine (CMBB)
      San Diego, CA, United States
  • 2002
    • Andhra University
      • School of Chemistry
      Vishākhapatnam, State of Andhra Pradesh, India
  • 1993
    • Chulalongkorn University
      • Department of Chemistry
      Bangkok, Bangkok, Thailand
    • Honolulu University
      Honolulu, Hawaii, United States
  • 1992
    • Shizuoka University
      • Department of Applied Biological Chemistry
      Shizuoka-shi, Shizuoka-ken, Japan
    • CSU Mentor
      Long Beach, California, United States
  • 1990–1991
    • University of Illinois at Chicago
      • College of Pharmacy
      Chicago, IL, United States
  • 1985
    • University of Karachi
      • HEJ Research Institute of Chemistry
      Karachi, Sindh, Pakistan