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1.  Baulamycins A and B, Broad-Spectrum Antibiotics Identified as Inhibitors of Siderophore Biosynthesis in Staphylococcus aureus and Bacillus anthracis 
Siderophores are high-affinity iron chelators produced by microorganisms and frequently contribute to the virulence of human pathogens. Targeted inhibition of the biosynthesis of siderophores staphyloferrin B of Staphylococcus aureus and petrobactin of Bacillus anthracis hold considerable potential as a single or combined treatment for methicillin-resistant S. aureus (MRSA) and anthrax infection, respectively. The biosynthetic pathways for both siderophores involve a nonribosomal peptide synthetase independent siderophore (NIS) synthetase, including SbnE in staphyloferrin B and AsbA in petrobactin. In this study, we developed a biochemical assay specific for NIS synthetases to screen for inhibitors of SbnE and AsbA against a library of marine microbial-derived natural product extracts (NPEs). Analysis of the NPE derived from Streptomyces tempisquensis led to the isolation of the novel antibiotics baulamycins A (BmcA, 6) and B (BmcB, 7). BmcA and BmcB displayed in vitro activity with IC50 values of 4.8 µM and 19 µM against SbnE and 180 µM and 200 µM against AsbA, respectively. Kinetic analysis showed that the compounds function as reversible competitive enzyme inhibitors. Liquid culture studies with S. aureus, B. anthracis, E. coli and several other bacterial pathogens demonstrated the capacity of these natural products to penetrate bacterial barriers and inhibit growth of both Gram-positive and Gram-negative species. These studies provide proof-of-concept that natural product inhibitors targeting siderophore virulence factors can provide access to novel broad-spectrum antibiotics, which may serve as important leads for the development of potent anti-infective agents
PMCID: PMC4028973  PMID: 24401083
high throughput screening; Staphylococcus aureus; Bacillus anthracis; natural product; antibiotic
2.  Uncovering the Cultivable Microbial Diversity of Costa Rican Beetles and Its Ability to Break Down Plant Cell Wall Components 
PLoS ONE  2014;9(11):e113303.
Coleopterans are the most diverse insect order described to date. These organisms have acquired an array of survival mechanisms through their evolution, including highly efficient digestive systems. Therefore, the coleopteran intestinal microbiota constitutes an important source of novel plant cell wall-degrading enzymes with potential biotechnological applications. We isolated and described the cultivable fungi, actinomycetes and aerobic eubacteria associated with the gut of larvae and adults from six different beetle families colonizing decomposing logs in protected Costa Rican ecosystems. We obtained 611 isolates and performed phylogenetic analyses using the ITS region (fungi) and 16S rDNA (bacteria). The majority of fungal isolates belonged to the order Hypocreales (26% of 169 total), while the majority of actinomycetes belonged to the genus Streptomyces (86% of 241 total). Finally, we isolated 201 bacteria spanning 19 different families belonging into four phyla: Firmicutes, α, β and γ-proteobacteria. Subsequently, we focused on microbes isolated from Passalid beetles to test their ability to degrade plant cell wall polymers. Highest scores in these assays were achieved by a fungal isolate (Anthostomella sp.), two Streptomyces and one Bacillus bacterial isolates. Our study demonstrates that Costa Rican beetles harbor several types of cultivable microbes, some of which may be involved in symbiotic relationships that enable the insect to digest complex polymers such as lignocellulose.
PMCID: PMC4239062  PMID: 25411842
4.  Phenolic Compounds as Antiangiogenic CMG2 Inhibitors from Costa Rican Endophytic Fungi 
Targeting and inhibiting CMG2 (Capillary Morphogenesis Gene protein 2) represents a new strategy for therapeutic agents for cancer and retinal diseases due to CMG2’s role in blood vessel growth (angiogenesis). A high throughput FRET (Förster Resonance Energy Transfer) assay was developed for the identification of CMG2 inhibitors as anti-angiogenetic agents. Bioassay-guided separation led to the isolation and identification of two new compounds (1 and 2) from CR252M, an endophytic fungus Coccomyces proteae collected from a Costa Rican rainforest, and one known compound (3) from CR1207B (Aurapex penicillata). Secondary in vitro assays indicated anti-angiogenic activity. Compound 3 inhibited the endothelial cell migration at 52 µM, but did not show any endothelial cell antiproliferative effect at 156 µM. The structure of the two new compounds, A (1) and B (2), were elucidated on the basis of extensive spectroscopic analysis, including 1D and 2D NMR experiments.
PMCID: PMC3563091  PMID: 22910038
Fungus; Coccomyces proteae; Aurapex penicillata; CMG2; Phenolic
5.  Identification of Anziaic Acid, a Lichen Depside from Hypotrachyna sp., as a New Topoisomerase Poison Inhibitor 
PLoS ONE  2013;8(4):e60770.
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.
PMCID: PMC3620467  PMID: 23593306
6.  Titration-based screening for evaluation of natural product extracts: identification of an aspulvinone family of luciferase inhibitors 
Chemistry & biology  2011;18(11):1442-1452.
The chemical diversity of nature has tremendous potential for discovery of new molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, macro- and microorganisms has curtailed their use in lead discovery efforts. Here we describe a process for leveraging the concentration-response curves (CRCs) obtained from quantitative HTS to improve the initial selection of “actives” from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm aims to improve the probability that labor-intensive subsequent steps of re-culturing, extraction and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by x-ray crystallography.
PMCID: PMC3225805  PMID: 22118678
7.  Complementary Cell-Based High Throughput Screens Identify Novel Modulators of the Unfolded Protein Response 
Journal of Biomolecular Screening  2011;16(8):825-835.
Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than two decades indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, we hypothesized that high throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small molecule activators of the apoptotic arm of the UPR to control or kill OSCC. We have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR sub-pathways. A ~66K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of pre-fractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80μM. A series of citrinin derivatives were isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds we examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, we found that patulin at 2.5 – 10μM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34 and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.
PMCID: PMC3374590  PMID: 21844328
unfolded protein response; endoplasmic reticulum stress; cell-based assay; luciferase reporter; natural products
8.  Study of the diversity of culturable actinomycetes in the North Pacific and Caribbean coasts of Costa Rica 
Antonie van Leeuwenhoek  2009;96(1):71-78.
In this study, 137 actinomycetes were isolated from subtidal marine sediments in the North Pacific and Caribbean coasts of Costa Rica. Bioinformatics analysis of the 16S rRNA gene sequences assigned the isolates to 15 families and 21 genera. Streptomyces was the dominant genus while the remaining 20 genera were poorly represented. Nearly 70% of the phylotypes presented a coastal-restricted distribution whereas the other 30% were common inhabitants of both shores. The coastal tropical waters of Costa Rica showed a high diversity of actinomycetes, both in terms of the number of species and phylogenetic composition, although significant differences were observed between and within shores. The observed pattern of species distribution might be the result of several factors including the characteristics of the ecosystems, presence of endemic species and the influence of terrestrial runoff.
PMCID: PMC3065112  PMID: 19365710
Actinomycetes; Microbial Ecology; Cahuita; Gandoca-Manzanillo; Las Baulas; Cabo Blanco
9.  The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific 
PLoS Biology  2007;5(3):e77.
The world's oceans contain a complex mixture of micro-organisms that are for the most part, uncharacterized both genetically and biochemically. We report here a metagenomic study of the marine planktonic microbiota in which surface (mostly marine) water samples were analyzed as part of the Sorcerer II Global Ocean Sampling expedition. These samples, collected across a several-thousand km transect from the North Atlantic through the Panama Canal and ending in the South Pacific yielded an extensive dataset consisting of 7.7 million sequencing reads (6.3 billion bp). Though a few major microbial clades dominate the planktonic marine niche, the dataset contains great diversity with 85% of the assembled sequence and 57% of the unassembled data being unique at a 98% sequence identity cutoff. Using the metadata associated with each sample and sequencing library, we developed new comparative genomic and assembly methods. One comparative genomic method, termed “fragment recruitment,” addressed questions of genome structure, evolution, and taxonomic or phylogenetic diversity, as well as the biochemical diversity of genes and gene families. A second method, termed “extreme assembly,” made possible the assembly and reconstruction of large segments of abundant but clearly nonclonal organisms. Within all abundant populations analyzed, we found extensive intra-ribotype diversity in several forms: (1) extensive sequence variation within orthologous regions throughout a given genome; despite coverage of individual ribotypes approaching 500-fold, most individual sequencing reads are unique; (2) numerous changes in gene content some with direct adaptive implications; and (3) hypervariable genomic islands that are too variable to assemble. The intra-ribotype diversity is organized into genetically isolated populations that have overlapping but independent distributions, implying distinct environmental preference. We present novel methods for measuring the genomic similarity between metagenomic samples and show how they may be grouped into several community types. Specific functional adaptations can be identified both within individual ribotypes and across the entire community, including proteorhodopsin spectral tuning and the presence or absence of the phosphate-binding gene PstS.
Author Summary
Marine microbes remain elusive and mysterious, even though they are the most abundant life form in the ocean, form the base of the marine food web, and drive energy and nutrient cycling. We know so little about the vast majority of microbes because only a small percentage can be cultivated and studied in the lab. Here we report on the Global Ocean Sampling expedition, an environmental metagenomics project that aims to shed light on the role of marine microbes by sequencing their DNA without first needing to isolate individual organisms. A total of 41 different samples were taken from a wide variety of aquatic habitats collected over 8,000 km. The resulting 7.7 million sequencing reads provide an unprecedented look at the incredible diversity and heterogeneity in naturally occurring microbial populations. We have developed new bioinformatic methods to reconstitute large portions of both cultured and uncultured microbial genomes. Organism diversity is analyzed in relation to sampling locations and environmental pressures. Taken together, these data and analyses serve as a foundation for greatly expanding our understanding of individual microbial lineages and their evolution, the nature of marine microbial communities, and how they are impacted by and impact our world.
TheSorcerer II GOS expedition, data sampling, and analysis is described. The immense diversity in the sequence data required novel comparative genomic assembly methods, which uncovered genomic differences that marker-based methods could not.
PMCID: PMC1821060  PMID: 17355176

Results 1-9 (9)