The antibiotic resistant threat is continuing to grow, due in part to the overuse of antibiotics ... more The antibiotic resistant threat is continuing to grow, due in part to the overuse of antibiotics in livestock feed. Many nations in Europe have banned the use of antibiotics in feed, leading to higher rates of infection in livestock animals and reduced productivity for the food market. Increasingly, researchers are looking into the efficacy of phytopreparations to replace antibiotics in feed, allowing for increased animal health without the development of resistance. Macleaya cordata, or Chinese plume poppy, shows promise as a food additive. To evaluate the antimicrobial efficacy of this plant, we tested in vitro activity of M. cordata extract, as well as pure compounds sanguinarine and chelerythrine against wild-type, methicillin-resistant, and multiply-resistant strains of Staphylococcus aureus (SA1199, AH1263, and IA116, respectively). Combination tests to evaluate synergy, additivity, and antagonism within the extract were also completed for the first time. Sanguinarine and chelerythrine showed complete growth inhibition of all strains of S. aureus at concentrations ranging from 3-10 µg/mL, and were equal in activity or were more potent than the reference compound chloramphenicol. Combination studies of pure sanguinarine and chelerythrine with M. cordata extract revealed additivity or indifference of mixture components with these compounds. Because sanguinarine and chelerythrine represent the major active constituents of M. cordata, the pooled amounts of these two compounds may be useful for establishing potency for quality control purposes. This is the first report of activity of chelerythrine and sanguinarine against methicillin-resistant S. aureus AH1263 and multiplyresistant S. aureus IA116, and illustrates the promise of M. cordata extract as an alternative to antibiotics in feed additives.
Proceedings of the National Academy of Sciences of the United States of America, May 3, 2021
Significance Fungi represent an underexploited resource for new compounds with applications in th... more Significance Fungi represent an underexploited resource for new compounds with applications in the pharmaceutical and agriscience industries. Despite the availability of >1,000 fungal genomes, our knowledge of the biosynthetic space encoded by these genomes is limited and ad hoc. We present results from systematically organizing the biosynthetic content of 1,037 fungal genomes, providing a resource for data-driven genome mining and large-scale comparison of the genetic and molecular repertoires produced in fungi, and compare to those present in bacteria.
Zika virus (ZIKV) is a dangerous human pathogen and no antiviral drugs have been approved to date... more Zika virus (ZIKV) is a dangerous human pathogen and no antiviral drugs have been approved to date. The chalcones are a group of small molecules that are found in a number of different plants, including Angelica keiskei Koidzumi, also known as ashitaba. To examine chalcone anti-ZIKV activity, three chalcones, 4-hydroxyderricin (4HD), xanthoangelol (XA), and xanthoangelol-E (XA-E), were purified from a methanol-ethyl acetate extract from A. keiskei. Molecular and ensemble docking predicted that these chalcones would establish multiple interactions with residues in the catalytic and allosteric sites of ZIKV NS2B-NS3 protease, and in the allosteric site of the NS5 RNA-dependent RNA-polymerase (RdRp). Machine learning models also predicted 4HD, XA and XA-E as potential anti-ZIKV inhibitors. Enzymatic and kinetic assays confirmed chalcone inhibition of the ZIKV NS2B-NS3 protease allosteric site with IC50s from 18 to 50 µM. Activity assays also revealed that XA, but not 4HD or XA-E, inhibited the allosteric site of the RdRp, with an IC50 of 6.9 µM. Finally, we tested these chalcones for their anti-viral activity in vitro with Vero cells. 4HD and XA-E displayed anti-ZIKV activity with EC50 values of 6.6 and 22.0 µM, respectively, while XA displayed relatively weak anti-ZIKV activity with whole cells. With their simple structures and relative ease of modification, the chalcones represent attractive candidates for hit-to-lead optimization in the search of new anti-ZIKV therapeutics.
Hydrastis canadensis, commonly known as goldenseal, is a botanical native to the southeastern Uni... more Hydrastis canadensis, commonly known as goldenseal, is a botanical native to the southeastern United States that has been used for the treatment of infection. The activity of goldenseal is often attributed to the presence of alkaloids (cyclic, nitrogen-containing compounds) present within its roots. Chemical components of botanical supplements like goldenseal may face degradation if not stored properly. The purpose of the research was to analyze the stability of known and unknown metabolites of H. canadensis during exposure to different storage conditions using mass spectrometry. Three abundant metabolites of H. canadensis, berberine, canadine, and hydrastine, were chosen for targeted analysis, and the stability of unknown metabolites was evaluated using untargeted metabolomics. The analysis and evaluation of H. canadensis samples were performed utilizing LC-MS and Principal Component Analysis (PCA). The research project focused on identifying the chemical changes in the metabolite ...
Hellebores (Helleborus spp.) are winter-flowering ornamental plants that are difficult to propaga... more Hellebores (Helleborus spp.) are winter-flowering ornamental plants that are difficult to propagate in tissue culture. To improve the performance of hellebores during micropropagation, preliminary tests were done to standardize temperature and light quality. The effects of growth hormone type and concentration on plant multiplication were also assessed. Performance remained low, and plantlets often displayed visible contamination. During Stage II micropropagation, plantlets of Helleborus ×nigercors were observed to be bacterially contaminated. To assess the effects of contaminants on performance in vitro, bacteria resistant to surface sterilization were isolated and Gram stained. Polymerase Chain Reaction (PCR) and 16S rRNA sequencing were used to identify bacterial isolates H7G and H7S at the genus level, belonging to Paenibacillus and Luteibacter genera, respectively. Strain H7R had high sequence similarity to the Pseudomonas, Stenotrophomonas, and Lysobacter genera. Paenibacillus sp. H7G was screened using multiple antibiotic treatments, including streptomycin sulfate, gentamicin sulfate, and/or cefotaxime, to determine bactericidal doses. Treatments were bactericidal if they contained at least 12.5 μg/mL gentamicin sulfate. Minimal bactericidal concentrations (MBCs) were unsuccessful at penetrating plant tissue, with none of treated plants being bacteria-free. When MBCs were quadrupled, severe phytotoxicity of plant tissues was noted, and viable bacteria remained. v ACKNOWLEDGMENTS I would like to express my special appreciation to my advisor, Dr. Jeffrey Adelberg, who has guided me through this project and allowed me to become the scientist I am today. I am grateful for his confidence in me, for it permitted me to gain respect for myself and my abilities. A great thank you to Jacqueline Naylor-Adelberg, who provided a clean and organized lab space and much-needed conversation. I would also like to thank Dr. Lissa Hayes for her contributions and advice on my research.
During micropropagation of Helleborus ×nigercors, plantlets were observed to be bacterially conta... more During micropropagation of Helleborus ×nigercors, plantlets were observed to be bacterially contaminated. To determine the identity of contaminants, bacteria resistant to surface sterilization were isolated and Gram stained. Polymerase Chain Reaction (PCR) and 16S rRNA sequencing were used to identify bacterial isolates H7G and H7S as belonging to the Paenibacillus and Luteibacteri genera, respectively. Strain H7R had highest sequence similarity to the Pseudomonas, Stenotrophomonas, and Lysobacter genera. Strains H7R and H7S were unable to grow in the absence of plant tissue and other bacterial species. Paenibacillus sp. H7G was screened using combinations of antibiotics including streptomycin sulfate, gentamicin sulfate, and cefotaxime, and was only eradicated by concentrations of gentamicin sulfate above phytotoxic levels. This is the first documented exploration of bacterial endophytes associated with Helleborus species.
Here we provide a comprehensive guide for studying natural product biosynthesis using genomics, m... more Here we provide a comprehensive guide for studying natural product biosynthesis using genomics, metabolomics, and their integrated datasets. We emphasize integrated strategies and provide a critical outlook on remaining challenges in the field.
Fungi are prolific producers of natural products, compounds which have had a large societal impac... more Fungi are prolific producers of natural products, compounds which have had a large societal impact as pharmaceuticals, mycotoxins, and agrochemicals. Despite the availability of over 1000 fungal genomes and several decades of compound discovery efforts from fungi, the biosynthetic gene clusters (BGCs) encoded by these genomes and the associated chemical space have yet to be analyzed systematically. Here we provide detailed annotation and analyses of fungal biosynthetic and chemical space to enable genome mining and discovery of fungal natural products. Using 1037 genomes from species across the fungal kingdom (e.g., Ascomycota, Basidiomycota, and non-Dikarya taxa), 36,399 predicted BGCs were organized into a network of 12,067 gene cluster families (GCFs). Anchoring these GCFs with reference BGCs enabled automated annotation of 2,026 BGCs with predicted metabolite scaffolds. We performed parallel analyses of the chemical repertoire of Fungi, organizing 15,213 fungal compounds into 2,...
Advances in genome sequencing have revitalized natural product discovery efforts, revealing the u... more Advances in genome sequencing have revitalized natural product discovery efforts, revealing the untapped biosynthetic potential of fungi. While the volume of genomic data continues to expand, discovery efforts are slowed due to the time-consuming nature of experiments required to characterize new molecules. To direct efforts toward uncharacterized biosynthetic gene clusters most likely to encode novel chemical scaffolds, we took advantage of comparative metabolomics and heterologous gene expression using fungal artificial chromosomes (FACs). By linking mass spectral profiles with structural clues provided by FAC-encoded gene clusters, we targeted a compound originating from an unusual gene cluster containing an indoleamine 2,3-dioxygenase (IDO). With this approach, we isolate and characterize R and S forms of the new molecule terreazepine, which contains a novel chemical scaffold resulting from cyclization of the IDO-supplied kynurenine. The discovery of terreazepine illustrates tha...
Staphylococcus aureus can colonize the human host and cause a variety of superficial and invasive... more Staphylococcus aureus can colonize the human host and cause a variety of superficial and invasive infections. The success of S. aureus as a pathogen derives from its ability to modulate its virulence through the release, sensing of and response to cyclic signaling peptides. Here we provide, for the first time, evidence that S. aureus processes and secretes small linear peptides through a specialized pathway that converts a lipoprotein leader into an extracellular peptide signal. We have identified and confirmed the machinery for each step and demonstrate that the putative membrane metalloprotease Eep and the EcsAB transporter are required to complete the processing and secretion of the peptides. In addition, we have identified several linear peptides, including the interspecies signaling molecule staph-cAM373, that are dependent on this processing and secretion pathway. These findings are particularly important because multiple Gram-positive bacteria rely on small linear peptides to...
This report documents the cellular, molecular, and analytical methods used to identify combinatio... more This report documents the cellular, molecular, and analytical methods used to identify combination effects in complex natural product mixtures.
The antibiotic resistant threat is continuing to grow, due in part to the overuse of antibiotics ... more The antibiotic resistant threat is continuing to grow, due in part to the overuse of antibiotics in livestock feed. Many nations in Europe have banned the use of antibiotics in feed, leading to higher rates of infection in livestock animals and reduced productivity for the food market. Increasingly, researchers are looking into the efficacy of phytopreparations to replace antibiotics in feed, allowing for increased animal health without the development of resistance. Macleaya cordata, or Chinese plume poppy, shows promise as a food additive. To evaluate the antimicrobial efficacy of this plant, we tested in vitro activity of M. cordata extract, as well as pure compounds sanguinarine and chelerythrine against wild-type, methicillin-resistant, and multiply-resistant strains of Staphylococcus aureus (SA1199, AH1263, and IA116, respectively). Combination tests to evaluate synergy, additivity, and antagonism within the extract were also completed for the first time. Sanguinarine and chelerythrine showed complete growth inhibition of all strains of S. aureus at concentrations ranging from 3-10 µg/mL, and were equal in activity or were more potent than the reference compound chloramphenicol. Combination studies of pure sanguinarine and chelerythrine with M. cordata extract revealed additivity or indifference of mixture components with these compounds. Because sanguinarine and chelerythrine represent the major active constituents of M. cordata, the pooled amounts of these two compounds may be useful for establishing potency for quality control purposes. This is the first report of activity of chelerythrine and sanguinarine against methicillin-resistant S. aureus AH1263 and multiplyresistant S. aureus IA116, and illustrates the promise of M. cordata extract as an alternative to antibiotics in feed additives.
Proceedings of the National Academy of Sciences of the United States of America, May 3, 2021
Significance Fungi represent an underexploited resource for new compounds with applications in th... more Significance Fungi represent an underexploited resource for new compounds with applications in the pharmaceutical and agriscience industries. Despite the availability of >1,000 fungal genomes, our knowledge of the biosynthetic space encoded by these genomes is limited and ad hoc. We present results from systematically organizing the biosynthetic content of 1,037 fungal genomes, providing a resource for data-driven genome mining and large-scale comparison of the genetic and molecular repertoires produced in fungi, and compare to those present in bacteria.
Zika virus (ZIKV) is a dangerous human pathogen and no antiviral drugs have been approved to date... more Zika virus (ZIKV) is a dangerous human pathogen and no antiviral drugs have been approved to date. The chalcones are a group of small molecules that are found in a number of different plants, including Angelica keiskei Koidzumi, also known as ashitaba. To examine chalcone anti-ZIKV activity, three chalcones, 4-hydroxyderricin (4HD), xanthoangelol (XA), and xanthoangelol-E (XA-E), were purified from a methanol-ethyl acetate extract from A. keiskei. Molecular and ensemble docking predicted that these chalcones would establish multiple interactions with residues in the catalytic and allosteric sites of ZIKV NS2B-NS3 protease, and in the allosteric site of the NS5 RNA-dependent RNA-polymerase (RdRp). Machine learning models also predicted 4HD, XA and XA-E as potential anti-ZIKV inhibitors. Enzymatic and kinetic assays confirmed chalcone inhibition of the ZIKV NS2B-NS3 protease allosteric site with IC50s from 18 to 50 µM. Activity assays also revealed that XA, but not 4HD or XA-E, inhibited the allosteric site of the RdRp, with an IC50 of 6.9 µM. Finally, we tested these chalcones for their anti-viral activity in vitro with Vero cells. 4HD and XA-E displayed anti-ZIKV activity with EC50 values of 6.6 and 22.0 µM, respectively, while XA displayed relatively weak anti-ZIKV activity with whole cells. With their simple structures and relative ease of modification, the chalcones represent attractive candidates for hit-to-lead optimization in the search of new anti-ZIKV therapeutics.
Hydrastis canadensis, commonly known as goldenseal, is a botanical native to the southeastern Uni... more Hydrastis canadensis, commonly known as goldenseal, is a botanical native to the southeastern United States that has been used for the treatment of infection. The activity of goldenseal is often attributed to the presence of alkaloids (cyclic, nitrogen-containing compounds) present within its roots. Chemical components of botanical supplements like goldenseal may face degradation if not stored properly. The purpose of the research was to analyze the stability of known and unknown metabolites of H. canadensis during exposure to different storage conditions using mass spectrometry. Three abundant metabolites of H. canadensis, berberine, canadine, and hydrastine, were chosen for targeted analysis, and the stability of unknown metabolites was evaluated using untargeted metabolomics. The analysis and evaluation of H. canadensis samples were performed utilizing LC-MS and Principal Component Analysis (PCA). The research project focused on identifying the chemical changes in the metabolite ...
Hellebores (Helleborus spp.) are winter-flowering ornamental plants that are difficult to propaga... more Hellebores (Helleborus spp.) are winter-flowering ornamental plants that are difficult to propagate in tissue culture. To improve the performance of hellebores during micropropagation, preliminary tests were done to standardize temperature and light quality. The effects of growth hormone type and concentration on plant multiplication were also assessed. Performance remained low, and plantlets often displayed visible contamination. During Stage II micropropagation, plantlets of Helleborus ×nigercors were observed to be bacterially contaminated. To assess the effects of contaminants on performance in vitro, bacteria resistant to surface sterilization were isolated and Gram stained. Polymerase Chain Reaction (PCR) and 16S rRNA sequencing were used to identify bacterial isolates H7G and H7S at the genus level, belonging to Paenibacillus and Luteibacter genera, respectively. Strain H7R had high sequence similarity to the Pseudomonas, Stenotrophomonas, and Lysobacter genera. Paenibacillus sp. H7G was screened using multiple antibiotic treatments, including streptomycin sulfate, gentamicin sulfate, and/or cefotaxime, to determine bactericidal doses. Treatments were bactericidal if they contained at least 12.5 μg/mL gentamicin sulfate. Minimal bactericidal concentrations (MBCs) were unsuccessful at penetrating plant tissue, with none of treated plants being bacteria-free. When MBCs were quadrupled, severe phytotoxicity of plant tissues was noted, and viable bacteria remained. v ACKNOWLEDGMENTS I would like to express my special appreciation to my advisor, Dr. Jeffrey Adelberg, who has guided me through this project and allowed me to become the scientist I am today. I am grateful for his confidence in me, for it permitted me to gain respect for myself and my abilities. A great thank you to Jacqueline Naylor-Adelberg, who provided a clean and organized lab space and much-needed conversation. I would also like to thank Dr. Lissa Hayes for her contributions and advice on my research.
During micropropagation of Helleborus ×nigercors, plantlets were observed to be bacterially conta... more During micropropagation of Helleborus ×nigercors, plantlets were observed to be bacterially contaminated. To determine the identity of contaminants, bacteria resistant to surface sterilization were isolated and Gram stained. Polymerase Chain Reaction (PCR) and 16S rRNA sequencing were used to identify bacterial isolates H7G and H7S as belonging to the Paenibacillus and Luteibacteri genera, respectively. Strain H7R had highest sequence similarity to the Pseudomonas, Stenotrophomonas, and Lysobacter genera. Strains H7R and H7S were unable to grow in the absence of plant tissue and other bacterial species. Paenibacillus sp. H7G was screened using combinations of antibiotics including streptomycin sulfate, gentamicin sulfate, and cefotaxime, and was only eradicated by concentrations of gentamicin sulfate above phytotoxic levels. This is the first documented exploration of bacterial endophytes associated with Helleborus species.
Here we provide a comprehensive guide for studying natural product biosynthesis using genomics, m... more Here we provide a comprehensive guide for studying natural product biosynthesis using genomics, metabolomics, and their integrated datasets. We emphasize integrated strategies and provide a critical outlook on remaining challenges in the field.
Fungi are prolific producers of natural products, compounds which have had a large societal impac... more Fungi are prolific producers of natural products, compounds which have had a large societal impact as pharmaceuticals, mycotoxins, and agrochemicals. Despite the availability of over 1000 fungal genomes and several decades of compound discovery efforts from fungi, the biosynthetic gene clusters (BGCs) encoded by these genomes and the associated chemical space have yet to be analyzed systematically. Here we provide detailed annotation and analyses of fungal biosynthetic and chemical space to enable genome mining and discovery of fungal natural products. Using 1037 genomes from species across the fungal kingdom (e.g., Ascomycota, Basidiomycota, and non-Dikarya taxa), 36,399 predicted BGCs were organized into a network of 12,067 gene cluster families (GCFs). Anchoring these GCFs with reference BGCs enabled automated annotation of 2,026 BGCs with predicted metabolite scaffolds. We performed parallel analyses of the chemical repertoire of Fungi, organizing 15,213 fungal compounds into 2,...
Advances in genome sequencing have revitalized natural product discovery efforts, revealing the u... more Advances in genome sequencing have revitalized natural product discovery efforts, revealing the untapped biosynthetic potential of fungi. While the volume of genomic data continues to expand, discovery efforts are slowed due to the time-consuming nature of experiments required to characterize new molecules. To direct efforts toward uncharacterized biosynthetic gene clusters most likely to encode novel chemical scaffolds, we took advantage of comparative metabolomics and heterologous gene expression using fungal artificial chromosomes (FACs). By linking mass spectral profiles with structural clues provided by FAC-encoded gene clusters, we targeted a compound originating from an unusual gene cluster containing an indoleamine 2,3-dioxygenase (IDO). With this approach, we isolate and characterize R and S forms of the new molecule terreazepine, which contains a novel chemical scaffold resulting from cyclization of the IDO-supplied kynurenine. The discovery of terreazepine illustrates tha...
Staphylococcus aureus can colonize the human host and cause a variety of superficial and invasive... more Staphylococcus aureus can colonize the human host and cause a variety of superficial and invasive infections. The success of S. aureus as a pathogen derives from its ability to modulate its virulence through the release, sensing of and response to cyclic signaling peptides. Here we provide, for the first time, evidence that S. aureus processes and secretes small linear peptides through a specialized pathway that converts a lipoprotein leader into an extracellular peptide signal. We have identified and confirmed the machinery for each step and demonstrate that the putative membrane metalloprotease Eep and the EcsAB transporter are required to complete the processing and secretion of the peptides. In addition, we have identified several linear peptides, including the interspecies signaling molecule staph-cAM373, that are dependent on this processing and secretion pathway. These findings are particularly important because multiple Gram-positive bacteria rely on small linear peptides to...
This report documents the cellular, molecular, and analytical methods used to identify combinatio... more This report documents the cellular, molecular, and analytical methods used to identify combination effects in complex natural product mixtures.
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