Jon Clardy

Harvard Medical School, Boston, Massachusetts, United States

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Publications (601)1801.08 Total impact

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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;
  • 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
  • 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; · 13.73 Impact Factor
  • Pierre Stallforth, Jon Clardy
    Proceedings of the National Academy of Sciences 02/2014; · 9.74 Impact Factor
  • ACS Synthetic Biology 01/2014;
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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; · 9.74 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
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    ABSTRACT: Biofilms are often associated with human bacterial infections, and the natural tolerance of biofilms to antibiotics challenges treatment. Compounds with antibiofilm activity could become useful adjuncts to antibiotic therapy. We used norspermidine, a natural trigger for biofilm disassembly in the developmental cycle of Bacillus subtilis , to develop guanidine and biguanide compounds with up to 20-fold increased potency in preventing biofilm formation and breaking down existing biofilms. These compounds also were active against pathogenic Staphylococcus aureus . An integrated approach involving structure-activity relationships, protonation constants, and crystal structure data on a focused synthetic library revealed that precise spacing of positively charged groups and the total charge at physiological pH distinguish potent biofilm inhibitors.
    Journal of the American Chemical Society 02/2013; 135(8):2927-30. · 10.68 Impact Factor
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    ABSTRACT: CMG2 is a transmembrane extracellular matrix binding protein that is also an anthrax toxin receptor. We have shown that high affinity CMG2 binders can inhibit angiogenesis and tumor growth. We recently described a high throughput FRET assay to identify CMG2 inhibitors. We now report the serendipitous discovery that PGG (1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose) is a CMG2 inhibitor with anti-angiogenic activity. PGG is a gallotannin produced by a variety of medicinal plants that exhibits a wide variety of anti-tumor and other activities. We find that PGG inhibits CMG2 with a submicromolar IC50 and it also inhibits the migration of human dermal microvascular endothelial cells at similar concentrations in vitro. Finally, oral or intraperitoneal administration of PGG inhibits angiogenesis in the mouse corneal micropocket assay in vivo. Together, these results suggest that a portion of the in vivo anti-tumor activity of PGG may be the result of antiangiogenic activity mediated by inhibition of CMG2.
    Journal of Medicinal Chemistry 02/2013; · 5.61 Impact Factor
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    ABSTRACT: Topoisomerase inhibitors are effective for antibacterial and anticancer therapy because they can lead to the accumulation of the intermediate DNA cleavage complex formed by the topoisomerase enzymes, which trigger cell death. Here we report the application of a novel enzyme-based high-throughput screening assay to identify natural product extracts that can lead to increased accumulation of the DNA cleavage complex formed by recombinant Yersinia pestis topoisomerase I as part of a larger effort to identify new antibacterial compounds. Further characterization and fractionation of the screening positives from the primary assay led to the discovery of a depside, anziaic acid, from the lichen Hypotrachyna sp. as an inhibitor for both Y. pestis and Escherichia coli topoisomerase I. In in vitro assays, anziaic acid exhibits antibacterial activity against Bacillus subtilis and a membrane permeable strain of E. coli. Anziaic acid was also found to act as an inhibitor of human topoisomerase II but had little effect on human topoisomerase I. This is the first report of a depside with activity as a topoisomerase poison inhibitor and demonstrates the potential of this class of natural products as a source for new antibacterial and anticancer compounds.
    PLoS ONE 01/2013; 8(4):e60770. · 3.73 Impact Factor
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    Emily R Derbyshire, Jon Clardy
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    ABSTRACT: A chemoproteomics approach has been employed to identify a kinase that could be used as a druggable target in efforts to develop new treatments for African sleeping sickness.
    eLife Sciences 01/2013; 2:e01042.
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    ABSTRACT: In sexually reproducing animals, mating is essential for transmitting genetic information to the next generation and therefore animals have evolved mechanisms for optimizing the chance of successful mate location. In the soil nematode C. elegans, males approach hermaphrodites via the ascaroside pheromones, recognize hermaphrodites when their tails contact the hermaphrodites' body, and eventually mate with them. These processes are mediated by sensory signals specialized for sexual communication, but other mechanisms may also be used to optimize mate location. Here we describe associative learning whereby males use sodium chloride as a cue for hermaphrodite location. Both males and hermaphrodites normally avoid sodium chloride after associative conditioning with salt and starvation. However, we found that males become attracted to sodium chloride after conditioning with salt and starvation if hermaphrodites are present during conditioning. For this conditioning, which we call sexual conditioning, hermaphrodites are detected by males through pheromonal signaling and additional cue(s). Sex transformation experiments suggest that neuronal sex of males is essential for sexual conditioning. Altogether, these results suggest that C. elegans males integrate environmental, internal and social signals to determine the optimal strategy for mate location.
    PLoS ONE 01/2013; 8(7):e68676. · 3.73 Impact Factor

Publication Stats

7k Citations
1,801.08 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
    • Cheikh Anta Diop University, Dakar
      Dakar, Dakar, Senegal
  • 2008–2009
    • Broad Institute of MIT and Harvard
      Cambridge, Massachusetts, United States
  • 2006–2008
    • Howard Hughes Medical Institute
      Maryland, United States
  • 2007
    • The Rockefeller University
      • Laboratory of Genetically Encoded Small Molecules
      New York City, NY, United States
  • 2003–2006
    • University of California, Santa Cruz
      • Department of Chemistry & Biochemistry
      Santa Cruz, CA, United States
    • University of California, San Diego
      • Center for Marine Biotechnology and Biomedicine (CMBB)
      San Diego, CA, United States
  • 1980–2006
    • Cornell University
      • Department of Chemistry and Chemical Biology
      Ithaca, NY, United States
  • 1974–2004
    • Iowa State University
      • Department of Chemistry
      Ames, IA, United States
  • 2002
    • Andhra University
      • School of Chemistry
      Vishākhapatnam, State of Andhra Pradesh, India
  • 1993
    • Chulalongkorn University
      • Department of Chemistry
      Bangkok, Bangkok, Thailand
    • University of Central Florida
      • Department of Chemistry
      Orlando, FL, United States
  • 1992
    • Shizuoka University
      • Department of Applied Biological Chemistry
      Shizuoka-shi, Shizuoka-ken, Japan
    • CSU Mentor
      Long Beach, California, United States
  • 1989–1991
    • University of Illinois at Chicago
      • College of Pharmacy
      Chicago, IL, United States
  • 1985
    • University of Karachi
      • HEJ Research Institute of Chemistry
      Karachi, Sindh, Pakistan