Publications by authors named "Tim S Bugni"

95 Publications

Phenyl-Lactic Acid Is an Active Ingredient in Bactericidal Supernatants of Lactobacillus crispatus.

J Bacteriol 2021 Sep 8;203(19):e0036021. Epub 2021 Sep 8.

Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicagogrid.164971.c, Maywood, Illinois, USA.

Lactobacillus crispatus is a well-established probiotic with antimicrobial activity against pathogens across several niches of the human body generally attributed to the production of bacteriostatic molecules, including hydrogen peroxide and lactic acid. Here, we show that the cell-free supernatants of clinical isolates of L. crispatus harbor robust bactericidal activity. We further identify phenyl-lactic acid as a bactericidal compound with properties and a susceptibility range nearly identical to that of the cell-free supernatant. As such, we hypothesize that phenyl-lactic acid is a key active ingredient in L. crispatus supernatant. Although Lactobacillus crispatus is an established commensal microbe frequently used in probiotics, its protective role in the bladder microbiome has not been clarified. We report here that some urinary isolates of L. crispatus exhibit bactericidal activity, primarily due to its ability to excrete phenyl-lactic acid into its environment. Both cell-free supernatants of L. crispatus isolates and phenyl-lactic acid exhibit bactericidal activity against a wide range of pathogens, including several that are resistant to multiple antibiotics.
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http://dx.doi.org/10.1128/JB.00360-21DOI Listing
September 2021

Discovery of anti-infective adipostatins through bioactivity-guided isolation and heterologous expression of a type III polyketide synthase.

Bioorg Chem 2021 07 22;112:104925. Epub 2021 Apr 22.

Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, United States. Electronic address:

Antibiotic resistance and emerging viral pandemics have posed an urgent need for new anti-infective drugs. By screening our microbial extract library against the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the notorious ESKAPE pathogens, an active fraction was identified and purified, leading to an initial isolation of adipostatins A (1) and B (2). In order to diversify the chemical structures of adipostatins toward enhanced biological activities, a type III polyketide synthase was identified from the native producer, Streptomyces davawensis DSM101723, and was subsequently expressed in an E. coli host, resulting in the isolation of nine additional adipostatins 3-11, including two new analogs (9 and 11). The structures of 1-11 were established by HRMS, NMR, and chemical derivatization, including using a microgram-scale meta-chloroperoxybenzoic acid epoxidation-MS/MS analysis to unambiguously determine the double bond position in the alkyl chain. The present study discovered SARS-CoV-2 main protease inhibitory activity for the class of adipostatins for the first time. Several of the adipostatins isolated also exhibited antimicrobial activity against selected ESKAPE pathogens.
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http://dx.doi.org/10.1016/j.bioorg.2021.104925DOI Listing
July 2021

from Fungus Gardens of Fungus-Growing Ants Produces Antifungals That Inhibit the Specialized Parasite .

Appl Environ Microbiol 2021 06 25;87(14):e0017821. Epub 2021 Jun 25.

Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.

Within animal-associated microbiomes, the functional roles of specific microbial taxa are often uncharacterized. Here, we use the fungus-growing ant system, a model for microbial symbiosis, to determine the potential defensive roles of key bacterial taxa present in the ants' fungus gardens. Fungus gardens serve as an external digestive system for the ants, with mutualistic fungi in the genus converting the plant substrate into energy for the ants. The fungus garden is host to specialized parasitic fungi in the genus . Here, we examine the potential role of spp. that occur within ant fungus gardens in inhibiting We isolated members of the bacterial genera and from 50% of the 52 colonies sampled, indicating that members of the family are common inhabitants in the fungus gardens of a diverse range of fungus-growing ant genera. Using antimicrobial inhibition bioassays, we found that 28 out of 32 isolates inhibited at least one strain with a zone of inhibition greater than 1 cm. Genomic assessment of fungus garden-associated indicated that isolates with strong inhibition all belonged to the genus and contained biosynthetic gene clusters that encoded the production of two antifungals: burkholdine1213 and pyrrolnitrin. Organic extracts of cultured isolates confirmed that these compounds are responsible for antifungal activities that inhibit but, at equivalent concentrations, not spp. Overall, these new findings, combined with previous evidence, suggest that members of the fungus garden microbiome play an important role in maintaining the health and function of fungus-growing ant colonies. Many organisms partner with microbes to defend themselves against parasites and pathogens. Fungus-growing ants must protect spp., the fungal mutualist that provides sustenance for the ants, from a specialized fungal parasite, . The ants take multiple approaches, including weeding their fungus gardens to remove spores, as well as harboring spp., bacteria that produce antifungals that inhibit In addition, a genus of bacteria commonly found in fungus gardens, , is known to produce secondary metabolites that inhibit spp. In this study, we isolated spp. from fungus-growing ants, assessed the isolates' ability to inhibit spp., and identified two compounds responsible for inhibition. Our findings suggest that spp. are often found in fungus gardens, adding another possible mechanism within the fungus-growing ant system to suppress the growth of the specialized parasite .
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http://dx.doi.org/10.1128/AEM.00178-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231715PMC
June 2021

Antileishmanial macrolides from ant-associated Streptomyces sp. ISID311.

Bioorg Med Chem 2021 02 12;32:116016. Epub 2021 Jan 12.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, SP, Brazil. Electronic address:

Three antifungal macrolides cyphomycin (1), caniferolide C (2) and GT-35 (3) were isolated from Streptomyces sp. ISID311, a bacterial symbiont associated with Cyphomyrmex fungus-growing ants. The planar structures of these compounds were established by 1 and 2D NMR data and MS analysis. The relative configurations of 1-3 were established using Kishi's universal NMR database method, NOE/ROE analysis and coupling constants analysis assisted by comparisons with NMR data of related compounds. Detailed bioinformatic analysis of cyphomycin biosynthetic gene cluster confirmed the stereochemical assignments. Compounds 1-3 displayed high antagonism against different strains of Escovopsis sp., pathogen fungi specialized to the fungus-growing ant system. Compounds 1-3 also exhibited potent antiprotozoal activity against intracellular amastigotes of the human parasite Leishmania donovani with IC values of 2.32, 0.091 and 0.073 µM, respectively, with high selectivity indexes.
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http://dx.doi.org/10.1016/j.bmc.2021.116016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923958PMC
February 2021

Turbinmicin inhibits Candida biofilm growth by disrupting fungal vesicle-mediated trafficking.

J Clin Invest 2021 03;131(5)

Department of Medicine, Medical Microbiology and Immunology.

The emergence of drug-resistant fungi has prompted an urgent threat alert from the US Centers for Disease Control (CDC). Biofilm assembly by these pathogens further impairs effective therapy. We recently identified an antifungal, turbinmicin, that inhibits the fungal vesicle-mediated trafficking pathway and demonstrates broad-spectrum activity against planktonically growing fungi. During biofilm growth, vesicles with unique features play a critical role in the delivery of biofilm extracellular matrix components. As these components are largely responsible for the drug resistance associated with biofilm growth, we explored the utility of turbinmicin in the biofilm setting. We found that turbinmicin disrupted extracellular vesicle (EV) delivery during biofilm growth and that this impaired the subsequent assembly of the biofilm matrix. We demonstrated that elimination of the extracellular matrix rendered the drug-resistant biofilm communities susceptible to fungal killing by turbinmicin. Furthermore, the addition of turbinmicin to otherwise ineffective antifungal therapy potentiated the activity of these drugs. The underlying role of vesicles explains this dramatic activity and was supported by phenotype reversal with the addition of exogenous biofilm EVs. This striking capacity to cripple biofilm assembly mechanisms reveals a new approach to eradicating biofilms and sheds light on turbinmicin as a promising anti-biofilm drug.
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http://dx.doi.org/10.1172/JCI145123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919718PMC
March 2021

Structural and Biosynthetic Analysis of the Fabrubactins, Unusual Siderophores from Strain C58.

ACS Chem Biol 2021 01 29;16(1):125-135. Epub 2020 Dec 29.

Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.

Siderophores are iron-chelating molecules produced by microorganisms and plants to acquire exogenous iron. Siderophore biosynthetic enzymology often produces elaborate and unique molecules through unusual reactions to enable specific recognition by the producing organisms. Herein, we report the structure of two siderophore analogs from strain C58, which we named fabrubactin (FBN) A and FBN B. Additionally, we characterized the substrate specificities of the NRPS and PKS components. The structures suggest unique Favorskii-like rearrangements of the molecular backbone that we propose are catalyzed by the flavin-dependent monooxygenase, FbnE. FBN A and B contain a 1,1-dimethyl-3-amino-1,2,3,4-tetrahydro-7,8-dihydroxy-quinolin (Dmaq) moiety previously seen only in the anachelin cyanobacterial siderophores. We provide evidence that Dmaq is derived from l-DOPA and propose a mechanism for the formation of the mature Dmaq moiety. Our bioinformatic analyses suggest that FBN A and B and the anachelins belong to a large and diverse siderophore family widespread throughout the / group, α-proteobacteria, and cyanobacteria.
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http://dx.doi.org/10.1021/acschembio.0c00809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7882191PMC
January 2021

Bacillibactins E and F from a Marine Sponge-Associated sp.

J Nat Prod 2021 01 18;84(1):136-141. Epub 2020 Dec 18.

Pharmaceutical Sciences Division, University of Wisconsin-Madison, Madison, Wisconsin 53705, United States.

Chemical investigation of a marine sponge-associated sp. led to the discovery of bacillibactins E and F ( and ). Despite containing the well-established cyclic triester core of iron-binding natural products such as enterobactin, bacillibactins E and F ( and ) are the first bacterial siderophores that contain nicotinic and benzoic acid moieties. The structures of the new compounds, including their absolute configurations, were determined by extensive spectroscopic analyses and Marfey's method. A plausible biosynthetic pathway to and is proposed; this route bears great similarity to other previously established bacillibactin-like pathways but appears to differentiate itself by a promiscuous DhbE, which likely installs the nicotinic moiety of and the benzoic acid group of .
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http://dx.doi.org/10.1021/acs.jnatprod.0c01170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856188PMC
January 2021

A marine microbiome antifungal targets urgent-threat drug-resistant fungi.

Science 2020 11;370(6519):974-978

Pharmaceutical Sciences Division, University of Wisconsin-Madison, Madison, WI, USA.

New antifungal drugs are urgently needed to address the emergence and transcontinental spread of fungal infectious diseases, such as pandrug-resistant Leveraging the microbiomes of marine animals and cutting-edge metabolomics and genomic tools, we identified encouraging lead antifungal molecules with in vivo efficacy. The most promising lead, turbinmicin, displays potent in vitro and mouse-model efficacy toward multiple-drug-resistant fungal pathogens, exhibits a wide safety index, and functions through a fungal-specific mode of action, targeting Sec14 of the vesicular trafficking pathway. The efficacy, safety, and mode of action distinct from other antifungal drugs make turbinmicin a highly promising antifungal drug lead to help address devastating global fungal pathogens such as
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http://dx.doi.org/10.1126/science.abd6919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756952PMC
November 2020

Draft Genome Sequence of sp. Strain WMMC2535, a Marine Ascidian-Associated Bacterium.

Microbiol Resour Announc 2020 Aug 20;9(34). Epub 2020 Aug 20.

Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, Madison, Wisconsin, USA

WMMC2535, a representative of the myxobacteria (family ), was isolated from a ragged sea hare in the Florida Keys, and its genome was sequenced using PacBio technology. The WMMC2535 genome sequence is the first of this genus and validates the notion that myxobacteria represent outstanding sources of structurally diverse natural products.
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http://dx.doi.org/10.1128/MRA.00657-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441232PMC
August 2020

: Automated Hierarchical Clustering and Principal Component Analysis of Large Metabolomic Datasets in R.

Metabolites 2020 Jul 21;10(7). Epub 2020 Jul 21.

Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, Madison, WI 53705, USA.

Microbial natural product discovery programs face two main challenges today: rapidly prioritizing strains for discovering new molecules and avoiding the rediscovery of already known molecules. Typically, these problems have been tackled using biological assays to identify promising strains and techniques that model variance in a dataset such as PCA to highlight novel chemistry. While these tools have shown successful outcomes in the past, datasets are becoming much larger and require a new approach. Since PCA models are dependent on the members of the group being modeled, large datasets with many members make it difficult to accurately model the variance in the data. Our tool, , first groups strains based on the similarity of their chemical composition, and then applies PCA to the smaller sub-groups yielding more robust PCA models. This allows for scalable chemical comparisons among hundreds of strains with thousands of molecular features. As a proof of concept, we applied our open-source tool to a dataset with 1046 LCMS profiles of marine invertebrate associated bacteria and discovered three new analogs of an established anticancer agent from one promising strain.
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http://dx.doi.org/10.3390/metabo10070297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407629PMC
July 2020

Pyridine-2,6-Dithiocarboxylic Acid and Its Metal Complexes: New Inhibitors of New Delhi Metallo β-Lactamase-1.

Mar Drugs 2020 Jun 2;18(6). Epub 2020 Jun 2.

Pharmaceutical Sciences Division, University of Wisconsin-Madison, Madison, WI 53705, USA.

NDM-1 inhibitors; marine-derived sp.; carbapenem-resistant Enterobacteriaceae; metal chelators.
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http://dx.doi.org/10.3390/md18060295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374359PMC
June 2020

MS-Derived Isotopic Fine Structure Reveals Forazoline A as a Thioketone-Containing Marine-Derived Natural Product.

Org Lett 2020 02 4;22(4):1275-1279. Epub 2020 Feb 4.

Pharmaceutical Sciences Division , University of Wisconsin-Madison , Madison , Wisconsin 53705 , United States.

Forazoline A is a structurally complex PKS-NRPS hybrid produced by marine-derived sp. During the course of studies highlighting the application of IFS analysis as a powerful tool for natural products analysis, we were alerted to an earlier misinterpretation with respect to forazoline A structure elucidation. In particular, IFS reveals that forazoline A contains a thioketone moiety rarely seen in secondary metabolites and, thus, constitutes an even more intriguing structure than originally thought.
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http://dx.doi.org/10.1021/acs.orglett.9b04535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494057PMC
February 2020

Enhypyrazinones A and B, Pyrazinone Natural Products from a Marine-Derived Myxobacterium sp.

Mar Drugs 2019 Dec 12;17(12). Epub 2019 Dec 12.

Pharmaceutical Sciences Division, University of Wisconsin-Madison, Madison, WI 53705, USA.

To date, studies describing myxobacterial secondary metabolites have been relatively scarce in comparison to those addressing actinobacterial secondary metabolites. This realization suggests the immense potential of myxobacteria as an intriguing source of secondary metabolites with unusual structural features and a wide array of biological activities. Marine-derived myxobacteria are especially attractive due to their unique biosynthetic gene clusters, although they are more difficult to handle than terrestrial myxobacteria. Here, we report the discovery of two new pyrazinone-type molecules, enhypyrazinones A and B, from a marine-derived myxobacterium sp. Their structures were elucidated by HRESIMS and comprehensive NMR data analyses. Compounds and , which contain a rare trisubstituted-pyrazinone core, represent a unique class of molecules from sp.
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http://dx.doi.org/10.3390/md17120698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950740PMC
December 2019

Phallusialides A-E, Pyrrole-Derived Alkaloids Discovered from a Marine-Derived sp. Bacterium Using MS-Based Metabolomics Approaches.

J Nat Prod 2019 12 3;82(12):3432-3439. Epub 2019 Dec 3.

Pharmaceutical Sciences Division , University of Wisconsin-Madison , Madison , Wisconsin 53705 , United States.

Integrating MS-based metabolomics approaches, LC-MS-PCA and molecular networking enabled the targeted isolation of five new pyrrole-derived alkaloids, phallusialides A-E (-), from a marine-derived sp. bacterium. The structures of - were elucidated by analysis of their HRMS, MS/MS, and NMR spectroscopic data. The absolute configuration of phallusialide A () was determined on the basis of comparisons of experimental and theoretically calculated ECD spectra. Compounds and exhibited antibacterial activity against methicillin resistant (MRSA) and , with MIC values of 32 and 64 μg/mL, respectively, whereas - showed no antibacterial activity even at 256 μg/mL, yielding important SAR insights for this class of compounds.
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http://dx.doi.org/10.1021/acs.jnatprod.9b00808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7784719PMC
December 2019

Pyonitrins A-D: Chimeric Natural Products Produced by .

J Am Chem Soc 2019 10 16;141(43):17098-17101. Epub 2019 Oct 16.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School , 240 Longwood Avenue , Boston , Massachusetts 02115 , United States.

Bacterial symbionts frequently provide chemical defenses for their hosts, and such systems can provide discovery pathways to new antifungals and structurally intriguing metabolites. This report describes a small family of naturally occurring small molecules with chimeric structures and a mixed biosynthesis that features an unexpected but key nonenzymatic step. An insect-associated strain's activity in an murine candidiasis assay led to the discovery of a family of highly hydrogen-deficient metabolites. Bioactivity- and mass-guided fractionation led to the pyonitrins, highly complex aromatic metabolites in which 10 of the 20 carbons are quaternary, and 7 of them are contiguous. The genome revealed that the production of the pyonitrins is the result of a spontaneous reaction between biosynthetic intermediates of two well-studied metabolites, pyochelin and pyrrolnitrin. The combined discovery of the pyonitrins and identification of the responsible biosynthetic gene clusters revealed an unexpected biosynthetic route that would have prevented the discovery of these metabolites by bioinformatic analysis alone.
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http://dx.doi.org/10.1021/jacs.9b09739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823628PMC
October 2019

Antifungal compounds from Streptomyces associated with attine ants also inhibit Leishmania donovani.

PLoS Negl Trop Dis 2019 08 5;13(8):e0007643. Epub 2019 Aug 5.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.

Bacterial strains isolated from attine ants showed activity against the insect specialized fungal pathogen Escovopsis and also against the human protozoan parasite Leishmania donovani. The bioassay guided fractionation of extracts from cultures of Streptomyces sp. ICBG292, isolated from the exoskeleton of Cyphomyrmex workers, led to the isolation of Mer-A2026B (1), piericidin-A1 (2) and nigericin (3). Nigericin (3) presented high activity against intracellular amastigotes of L. donovani (IC50 0.129 ± 0.008 μM). Streptomyces puniceus ICBG378, isolated from workers of Acromyrmex rugosus rugosus, produced dinactin (4) with potent anti-L. donovani activity against intracellular amastigotes (IC50 0.018 ± 0.003 μM). Compounds 3 and 4 showed good selectivity indexes, 88.91 and 656.11 respectively, and were more active than positive control, miltefosine. Compounds 1-4 were also active against some Escovopsis strains. Compounds 1 and 2 were also produced by Streptomyces sp. ICBG233, isolated from workers of Atta sexdens, and detected in ants' extracts by mass spectrometry, suggesting they are produced in the natural environment as defensive compounds involved in the symbiotic interaction.
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http://dx.doi.org/10.1371/journal.pntd.0007643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695191PMC
August 2019

Madurastatin D1 and D2, Oxazoline Containing Siderophores Isolated from an

Org Lett 2019 08 5;21(16):6275-6279. Epub 2019 Aug 5.

Pharmaceutical Sciences Division , University of Wisconsin-Madison , 777 Highland Avenue , Madison , Wisconsin 53705 , United States.

Two new siderophores, madurastatin D1 and D2, together with (-)-madurastatin C1, the enantiomer of a known compound, were isolated from marine-derived sp. The presence of an unusual 4-imidazolidinone ring in madurastatins D1 and D2 inspired us to sequence the sp. genome and to identify the biosynthetic gene cluster, knowledge of which enables us to now propose a biosynthetic pathway. Madurastatin D1 and D2 are moderately active in antimicrobial assays with .
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http://dx.doi.org/10.1021/acs.orglett.9b02159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941472PMC
August 2019

Bulbiferates A and B: Antibacterial Acetamidohydroxybenzoates from a Marine Proteobacterium, sp.

J Nat Prod 2019 07 4;82(7):1930-1934. Epub 2019 Jun 4.

Pharmaceutical Sciences Division, School of Pharmacy , University of Wisconsin , Madison , Wisconsin 53705 , United States.

Here we report the discovery of two new 3-acetamido-4-hydroxybenzoate esters, bulbiferates A () and B (), isolated from sp. cultivated from the marine tunicate . The structures of and were determined by analysis of 2D NMR and MS data. Additionally, three synthetic analogues (-), differing in ester sizes/lengths, were prepared for the purposes of evaluating potential structure-activity relationships; no clear correlations tying ester lengths to activity were evident. Bulbiferates A () and B () demonstrated antibacterial activity against both () and methicillin-sensitive (MSSA), whereas the synthetic analogues and displayed activity only against MSSA.
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http://dx.doi.org/10.1021/acs.jnatprod.9b00312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660402PMC
July 2019

Omics Technologies to Understand Activation of a Biosynthetic Gene Cluster in Micromonospora sp. WMMB235: Deciphering Keyicin Biosynthesis.

ACS Chem Biol 2019 06 7;14(6):1260-1270. Epub 2019 Jun 7.

Pharmaceutical Sciences Division , University of Wisconsin-Madison , Madison , Wisconsin 53705 , United States.

DNA sequencing of a large collection of bacterial genomes reveals a wealth of orphan biosynthetic gene clusters (BGCs) with no identifiable products. BGC silencing, for those orphan clusters that are truly silent, rather than those whose products have simply evaded detection and cluster correlation, is postulated to result from transcriptional inactivation of these clusters under standard laboratory conditions. Here, we employ a multi-omics approach to demonstrate how interspecies interactions modulate the keyicin producing kyc cluster at the transcriptome level in cocultures of kyc-bearing Micromonospora sp. and a Rhodococcus sp. We further correlate coculture dependent changes in keyicin production to changes in transcriptomic and proteomic profiles and show that these changes are attributable to small molecule signaling consistent with a quorum sensing pathway. In piecing together the various elements underlying keyicin production in coculture, this study highlights how omics technologies can expedite future efforts to understand and exploit silent BGCs.
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http://dx.doi.org/10.1021/acschembio.9b00223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591704PMC
June 2019

The antimicrobial potential of Streptomyces from insect microbiomes.

Nat Commun 2019 01 31;10(1):516. Epub 2019 Jan 31.

Department of Bacteriology, University of Wisconsin-Madison, Madison, 53706, WI, USA.

Antimicrobial resistance is a global health crisis and few novel antimicrobials have been discovered in recent decades. Natural products, particularly from Streptomyces, are the source of most antimicrobials, yet discovery campaigns focusing on Streptomyces from the soil largely rediscover known compounds. Investigation of understudied and symbiotic sources has seen some success, yet no studies have systematically explored microbiomes for antimicrobials. Here we assess the distinct evolutionary lineages of Streptomyces from insect microbiomes as a source of new antimicrobials through large-scale isolations, bioactivity assays, genomics, metabolomics, and in vivo infection models. Insect-associated Streptomyces inhibit antimicrobial-resistant pathogens more than soil Streptomyces. Genomics and metabolomics reveal their diverse biosynthetic capabilities. Further, we describe cyphomycin, a new molecule active against multidrug resistant fungal pathogens. The evolutionary trajectories of Streptomyces from the insect microbiome influence their biosynthetic potential and ability to inhibit resistant pathogens, supporting the promise of this source in augmenting future antimicrobial discovery.
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http://dx.doi.org/10.1038/s41467-019-08438-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355912PMC
January 2019

Biemamides A-E, Inhibitors of the TGF-β Pathway That Block the Epithelial to Mesenchymal Transition.

Org Lett 2018 09 30;20(18):5529-5532. Epub 2018 Aug 30.

Pharmaceutical Sciences Division , University of Wisconsin-Madison , Madison , Wisconsin 53705 , United States.

Screening of a marine natural products library for inhibitors of TGF-β revealed five pyrimidinedione derivatives, biemamides A-E (1-5). The structures were determined by 2D NMR and HRMS experiments; absolute configurations were established by advanced Marfey's analysis and ECD calculations. Biemamides A-E specifically inhibited in vitro TGF-β induced epithelial to mesenchymal transition in NMuMG cells. Additionally, using Caenorhabditis elegans, selected biemmamides were found to influence in vivo developmental processes related to body size regulation in a dose-dependent manner.
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http://dx.doi.org/10.1021/acs.orglett.8b01871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207949PMC
September 2018

Draft Genome Sequence of Micromonospora sp. Strain WMMA1996, a Marine Sponge-Associated Bacterium.

Genome Announc 2018 Feb 22;6(8). Epub 2018 Feb 22.

Pharmaceutical Sciences Division, University of Wisconsin-Madison, Madison, Wisconsin, USA

sp. strain WMMA1996 was isolated in 2013 off the coast of the Florida Keys, United States, from a marine sponge as part of bacterial coculture-based drug discovery initiatives. Analysis of the ∼6.44-Mb genome reveals this microbe's potential role in the discovery of new drugs.
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http://dx.doi.org/10.1128/genomeA.00077-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823995PMC
February 2018

Complete Genome Sequence of sp. Strain WMMA184, a Marine Coral-Associated Bacterium.

Genome Announc 2018 Feb 1;6(5). Epub 2018 Feb 1.

Pharmaceutical Sciences Division, University of Wisconsin-Madison, Madison, Wisconsin, USA

sp. strain WMMA184 was isolated from the marine coral as part of ongoing drug discovery efforts. Analysis of the 4.16-Mb genome provides information regarding interspecies interactions as it pertains to the regulation of secondary metabolism and natural product biosynthesis potential.
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http://dx.doi.org/10.1128/genomeA.01582-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5794961PMC
February 2018

Accessing chemical diversity from the uncultivated symbionts of small marine animals.

Nat Chem Biol 2018 02 1;14(2):179-185. Epub 2018 Jan 1.

Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah, USA.

Chemistry drives many biological interactions between the microbiota and host animals, yet it is often challenging to identify the chemicals involved. This poses a problem, as such small molecules are excellent sources of potential pharmaceuticals, pretested by nature for animal compatibility. We discovered anti-HIV compounds from small, marine tunicates from the Eastern Fields of Papua New Guinea. Tunicates are a reservoir for new bioactive chemicals, yet their small size often impedes identification or even detection of the chemicals within. We solved this problem by combining chemistry, metagenomics, and synthetic biology to directly identify and synthesize the natural products. We show that these anti-HIV compounds, the divamides, are a novel family of lanthipeptides produced by symbiotic bacteria living in the tunicate. Neighboring animal colonies contain structurally related divamides that differ starkly in their biological properties, suggesting a role for biosynthetic plasticity in a native context wherein biological interactions take place.
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http://dx.doi.org/10.1038/nchembio.2537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771842PMC
February 2018

Coculture of Marine Invertebrate-Associated Bacteria and Interdisciplinary Technologies Enable Biosynthesis and Discovery of a New Antibiotic, Keyicin.

ACS Chem Biol 2017 12 22;12(12):3093-3102. Epub 2017 Nov 22.

Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin , Madison, Wisconsin 53705, United States.

Advances in genomics and metabolomics have made clear in recent years that microbial biosynthetic capacities on Earth far exceed previous expectations. This is attributable, in part, to the realization that most microbial natural product (NP) producers harbor biosynthetic machineries not readily amenable to classical laboratory fermentation conditions. Such "cryptic" or dormant biosynthetic gene clusters (BGCs) encode for a vast assortment of potentially new antibiotics and, as such, have become extremely attractive targets for activation under controlled laboratory conditions. We report here that coculturing of a Rhodococcus sp. and a Micromonospora sp. affords keyicin, a new and otherwise unattainable bis-nitroglycosylated anthracycline whose mechanism of action (MOA) appears to deviate from those of other anthracyclines. The structure of keyicin was elucidated using high resolution MS and NMR technologies, as well as detailed molecular modeling studies. Sequencing of the keyicin BGC (within the Micromonospora genome) enabled both structural and genomic comparisons to other anthracycline-producing systems informing efforts to characterize keyicin. The new NP was found to be selectively active against Gram-positive bacteria including both Rhodococcus sp. and Mycobacterium sp. E. coli-based chemical genomics studies revealed that keyicin's MOA, in contrast to many other anthracyclines, does not invoke nucleic acid damage.
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http://dx.doi.org/10.1021/acschembio.7b00688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973552PMC
December 2017

Thalassosamide, a Siderophore Discovered from the Marine-Derived Bacterium Thalassospira profundimaris.

J Nat Prod 2017 09 25;80(9):2551-2555. Epub 2017 Aug 25.

Small Molecule Screening & Synthesis Facility, UW Carbone Cancer Center , Madison, Wisconsin 53792, United States.

Here we describe the rapid identification and prioritization of novel active marine natural products using an improved dereplication strategy. During the course of our screening of marine natural product libraries, a new cyclic trihydroxamate compound, thalassosamide, was discovered from the α-proteobacterium Thalassospira profundimaris. Its structure was determined by 2D NMR and MS/MS experiments, and the absolute configuration of the lysine-derived units was established by Marfey's analysis, whereas that of C-9, 9', and 9″ was determined via the circular dichroism data of the [Rh(OCOCF)] complex and DFT NMR calculations. Thalassosamide showed moderate in vivo efficacy against Pseudomonas aeruginosa.
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http://dx.doi.org/10.1021/acs.jnatprod.7b00328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740872PMC
September 2017

Chemical Genomics, Structure Elucidation, and in Vivo Studies of the Marine-Derived Anticlostridial Ecteinamycin.

ACS Chem Biol 2017 09 26;12(9):2287-2295. Epub 2017 Jul 26.

Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.

A polyether antibiotic, ecteinamycin (1), was isolated from a marine Actinomadura sp., cultivated from the ascidian Ecteinascidia turbinata. C enrichment, high resolution NMR spectroscopy, and molecular modeling enabled elucidation of the structure of 1, which was validated on the basis of comparisons with its recently reported crystal structure. Importantly, ecteinamycin demonstrated potent activity against the toxigenic strain of Clostridium difficile NAP1/B1/027 (MIC = 59 ng/μL), as well as other toxigenic and nontoxigenic C. difficile isolates both in vitro and in vivo. Additionally, chemical genomics studies using Escherichia coli barcoded deletion mutants led to the identification of sensitive mutants such as trkA and kdpD involved in potassium cation transport and homeostasis supporting a mechanistic proposal that ecteinamycin acts as an ionophore antibiotic. This is the first antibacterial agent whose mechanism of action has been studied using E. coli chemical genomics. On the basis of these data, we propose ecteinamycin as an ionophore antibiotic that causes C. difficile detoxification and cell death via potassium transport dysregulation.
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http://dx.doi.org/10.1021/acschembio.7b00388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697710PMC
September 2017

Natural Product Discovery Using Planes of Principal Component Analysis in R (PoPCAR).

Metabolites 2017 Jul 13;7(3). Epub 2017 Jul 13.

Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, Madison, WI 53705, USA.

Rediscovery of known natural products hinders the discovery of new, unique scaffolds. Efforts have mostly focused on streamlining the determination of what compounds are known vs. unknown (dereplication), but an alternative strategy is to focus on what is different. Utilizing statistics and assuming that common actinobacterial metabolites are likely known, focus can be shifted away from dereplication and towards discovery. LC-MS-based principal component analysis (PCA) provides a perfect tool to distinguish unique vs. common metabolites, but the variability inherent within natural products leads to datasets that do not fit ideal standards. To simplify the analysis of PCA models, we developed a script that identifies only those masses or molecules that are unique to each strain within a group, thereby greatly reducing the number of data points to be inspected manually. Since the script is written in R, it facilitates integration with other metabolomics workflows and supports automated mass matching to databases such as Antibase.
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http://dx.doi.org/10.3390/metabo7030034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5618319PMC
July 2017
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