Herptile gut microbiomes: a natural system to study multi-kingdom interactions between filamentous fungi and bacteria.

Reptiles and amphibians (herptiles) are some of the most endangered and threatened species on the planet and numerous conservation strategies are being implemented with the goal of ensuring species recovery. Little is known, however, about the gut microbiome of wild herptiles and how it relates to the health of these populations. Here, we report results from the gut microbiome characterization of both a broad survey of herptiles, and the correlation between the fungus Basidiobolus, and the bacterial community supported by a deeper, more intensive sampling of Plethodon glutinosus, known as slimy salamanders. We demonstrate that bacterial communities sampled from frogs, lizards, and salamanders are structured by the host taxonomy and that Basidiobolus is a common and natural component of these wild gut microbiomes. Intensive sampling of multiple hosts across the ecoregions of Tennessee revealed that geography and host:geography interactions are strong predictors of distinct Basidiobolus operational taxonomic units present within a given host. Co-occurrence analyses of Basidiobolus and bacterial community diversity support a correlation and interaction between Basidiobolus and bacteria, suggesting that Basidiobolus may play a role in structuring the bacterial community. We further the hypothesis that this interaction is advanced by unique specialized metabolism originating from horizontal gene transfer from bacteria to Basidiobolus and demonstrate that Basidiobolus is capable of producing a diversity of specialized metabolites including small cyclic peptides.IMPORTANCEThis work significantly advances our understanding of biodiversity and microbial interactions in herptile microbiomes, the role that fungi play as a structural and functional members of herptile gut microbiomes, and the chemical functions that structure microbiome phenotypes. We also provide an important observational system of how the gut microbiome represents a unique environment that selects for novel metabolic functions through horizontal gene transfer between fungi and bacteria. Such studies are needed to better understand the complexity of gut microbiomes in nature and will inform conservation strategies for threatened species of herpetofauna.

New species and new combinations in the genus Paraisaria (Hypocreales, Ophiocordycipitaceae) from the U.S.A., supported by polyphasic analysis.

Molecular phylogenetic and chemical analyses, and morphological characterization of collections of North American Paraisaria specimens support the description of two new species and two new combinations for known species. P.cascadensissp. nov. is a pathogen of Cyphoderris (Orthoptera) from the Pacific Northwest USA and P.pseudoheteropodasp. nov. is a pathogen of cicadae (Hemiptera) from the Southeast USA. New combinations are made for Ophiocordycepsinsignis and O.monticola based on morphological, ecological, and chemical study. A new cyclopeptide family proved indispensable in providing chemotaxonomic markers for resolving species in degraded herbarium specimens for which DNA sequencing is intractable. This approach enabled the critical linkage of a 142-year-old type specimen to a phylogenetic clade. The diversity of Paraisaria in North America and the utility of chemotaxonomy for the genus are discussed.

Design of Synthetic Surrogates for the Macrolactone Linker Motif in Coibamide A.

A marine cyanobacterial cyclic depsipeptide, coibamide A (CbA), inhibits the mammalian protein secretory pathway by blocking the Sec61 translocon, which is an emerging drug target for cancer and other chronic diseases. In our previous structure-activity relationship study of CbA, the macrolactone ester linker was replaced with alkyl/alkenyl surrogates to provide synthetically accessible macrocyclic scaffolds. To optimize the cellular bioactivity profile of CbA analogues, novel lysine mimetics having ß- and ε-methyl groups have now been designed and synthesized by a stereoselective route. A significant increase in cytotoxicity was observed upon introduction of these two methyl groups, corresponding to the d-MeAla α-methyl and MeThr ß-methyl of CbA. All synthetic products retained the ability to inhibit secretion of a model Sec61 substrate. Tandem evaluation of secretory function inhibition in living cells and cytotoxicity was an effective strategy to assess the impact of structural modifications to the linker for ring closure.

Variations and gradients between methane seep and off-seep microbial communities in a submarine canyon system in the Northeast Pacific.

Methane seeps are highly abundant marine habitats that contribute sources of chemosynthetic primary production to marine ecosystems. Seeps also factor into the global budget of methane, a potent greenhouse gas. Because of these factors, methane seeps influence not only local ocean ecology, but also biogeochemical cycles on a greater scale. Methane seeps host specialized microbial communities that vary significantly based on geography, seep gross morphology, biogeochemistry, and a diversity of other ecological factors including cross-domain species interactions. In this study, we collected sediment cores from six seep and non-seep locations from Grays and Quinault Canyons (46-47°N) off Washington State, USA, as well as one non-seep site off the coast of Oregon, USA (45°N) to quantify the scale of seep influence on biodiversity within marine habitats. These samples were profiled using 16S rRNA gene sequencing. Predicted gene functions were generated using the program PICRUSt2, and the community composition and predicted functions were compared among samples. The microbial communities at seeps varied by seep morphology and habitat, whereas the microbial communities at non-seep sites varied by water depth. Microbial community composition and predicted gene function clearly transitioned from on-seep to off-seep in samples collected from transects moving away from seeps, with a clear ecotone and high diversity where methane-fueled habitats transition into the non-seep deep sea. Our work demonstrates the microbial and metabolic sphere of influence that extends outwards from methane seep habitats.

A Quarter Century of Marine Biodiscovery in Algoa Bay, South Africa.

Algoa Bay, the largest crenulate bay on the southeastern coast of South Africa, is currently one of the most well-studied marine ecosystems in southern Africa. A plethora of endemic marine invertebrates inhabits the benthic reefs on the western edge of the Bay in close proximity to South Africa's sixth largest city. Over the past 25 years, South African marine natural products chemists, together with international collaborators from the US National Cancer Institute and other US institutions, have focused their attention on Algoa Bay's benthic marine invertebrates as a potential source of new anticancer compounds. This review commemorates a quarter of a century of marine biodiscovery in Algoa Bay and presents the structures and bioactivities of 49 new and 36 known specialized metabolites isolated from two molluscs, eight ascidians, and six sponges. Thirty-nine of these compounds were cytotoxic to cancer cells in vitro with 20 exhibiting moderate to potent cytotoxicity. Six other compounds exhibited antimicrobial activity. Foremost among the potential anticancer compounds is mandelalide A (38) from the Algoa Bay ascidian Lissoclinum species.

Streptomyces spiramenti sp. nov., isolated from a deep-sea microbial mat.

Strain 5675061T was isolated from a deep-sea microbial mat near hydrothermal vents within the Axial Seamount caldera on the Juan de Fuca Ridge (NE Pacific Ocean) and was taxonomically evaluated using a polyphasic approach. Morphological and chemotaxonomic properties are consistent with characteristics of the genus Streptomyces: aerobic Gram-stain-positive filaments that form spores, L,L-diaminopimelic acid in whole-cell hydrolysates, and iso-C16:0 as the major fatty acid. Phylogenetic analysis, genomic, and biochemical comparisons show close evolutionary relatedness to Streptomyces lonarensis NCL716T, S. bohaiensis 11A07T, and S. otsuchiensis OTB305T but genomic relatedness indices identify strain 5675061T as a distinct species. Based on a polyphasic characterization, identifying differences in genomic and taxonomic data, strain 5675061T represents a novel species, for which the name Streptomyces spiramenti sp. nov. is proposed. The type strain is 5675061T (=LMG 31896T = DSM 111793T).

The Marine-Derived Macrolactone Mandelalide A Is an Indirect Activator of AMPK.

The mandelalides are complex macrolactone natural products with distinct macrocycle motifs and a bioactivity profile that is heavily influenced by compound glycosylation. Mandelalides A and B are direct inhibitors of mitochondrial ATP synthase (complex V) and therefore more toxic to mammalian cells with an oxidative metabolic phenotype. To provide further insight into the pharmacology of the mandelalides, we studied the AMP-activated protein kinase (AMPK) energy stress pathway and report that mandelalide A is an indirect activator of AMPK. Wild-type mouse embryonic fibroblasts (MEFs) and representative human non-small cell lung cancer (NSCLC) cells showed statistically significant increases in phospho-AMPK (Thr172) and phospho-ACC (Ser79) in response to mandelalide A. Mandelalide L, which also harbors an A-type macrocycle, induced similar increases in phospho-AMPK (Thr172) and phospho-ACC (Ser79) in U87-MG glioblastoma cells. In contrast, MEFs co-treated with an AMPK inhibitor (dorsomorphin), AMPKα-null MEFs, or NSCLC cells lacking liver kinase B1 (LKB1) lacked this activity. Mandelalide A was significantly more cytotoxic to AMPKα-null MEFs than wild-type cells, suggesting that AMPK activation serves as a protective response to mandelalide-induced depletion of cellular ATP. However, LKB1 status alone was not predictive of the antiproliferative effects of mandelalide A against NSCLC cells. When EGFR status was considered, erlotinib and mandelalide A showed strong cytotoxic synergy in combination against erlotinib-resistant 11-18 NSCLC cells but not against erlotinib-sensitive PC-9 cells. Finally, prolonged exposures rendered mandelalide A, a potent and efficacious cytotoxin, against a panel of human glioblastoma cell types regardless of the underlying metabolic phenotype of the cell. These results add biological relevance to the mandelalide series and provide the basis for their further pre-clinical evaluation as ATP synthase inhibitors and secondary activators of AMPK.

Tolypocladamide H and the Proposed Tolypocladamide NRPS in Tolypocladium Species.

The genome of entomopathogenic fungus Tolypocladium inflatum Gams encodes 43 putative biosynthetic gene clusters for specialized metabolites, although genotype-phenotype linkages have been reported only for the cyclosporins and fumonisins. T. inflatum was cultured in defined minimal media, supplemented with or without one of nine different amino acids. Acquisition of LC-MS/MS data for molecular networking and manual analysis facilitated annotation of putative known and unknown metabolites. These data led us to target a family of peptaibols and guided the isolation and purification of tolypocladamide H (1), which showed modest antibacterial activity and toxicity to mammalian cells at micromolar concentrations. HRMS/MS, NMR, and advanced Marfey's analysis were used to assign the structure of 1 as a peptaibol containing 4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt), a hallmark structural motif of the cyclosporins. LC-MS detection of homologous tolypocladamide metabolites and phylogenomic analyses of peptaibol biosynthetic genes in other cultured Tolypocladium species allowed assignment of a putative tolypocladamide nonribosomal peptide synthetase gene.

Canine osteosarcoma cells exhibit basal accumulation of multiple chaperone proteins and are sensitive to small molecule inhibitors of GRP78 and heat shock protein function.

Osteosarcoma is the most common type of bone cancer in dogs and humans, with significant numbers of patients experiencing treatment failure and disease progression. In our search for new approaches to treat osteosarcoma, we previously detected multiple chaperone proteins in the surface-exposed proteome of canine osteosarcoma cells. In the present study, we characterized expression of representative chaperones and find evidence for stress adaptation in canine osteosarcoma cells relative to osteogenic progenitors from normal bone. We compared the cytotoxic potential of direct (HA15) and putative (OSU-03012) inhibitors of Grp78 function and found canine POS and HMPOS osteosarcoma cells to be more sensitive to both compounds than normal cells. HA15 and OSU-03012 increased the thermal stability of Grp78 in intact POS cells at low micromolar concentrations, but each induced distinct patterns in Grp78 expression without significant change in Grp94. Both inhibitors were as effective alone as carboplatin and showed little evidence of synergy in combination treatment. However, HMPOS cells with acquired resistance to carboplatin were sensitive to inhibition of Grp78 (by HA15; OSU-03012), Hsp70 (by VER-155008), and Hsp90 (by 17-AAG) function. These results suggest that multiple nodes within the osteosarcoma chaperome may be relevant chemotherapeutic targets against platinum resistance.

Design of Coibamide A Mimetics with Improved Cellular Bioactivity.

Coibamide A, a cyclic depsipeptide isolated from a Panamanian marine cyanobacterium, shows potent cytotoxic activity via the inhibition of the Sec61 translocon. We designed a coibamide A mimetic in which the ester linkage between MeThr and d-MeAla in coibamide A was replaced with an alkyl linker to provide a stable macrocyclic scaffold possessing a MeLys(Me) residue. Taking advantage of a facile solid-phase synthetic approach, an structure-activity relationship (SAR) study of the newly designed macrocyclic structure was performed, with a focus on altering the pattern of N-methyl substitution and amino acid configurations. Overall, the simplified macrocyclic scaffold with an alkyl linker resulted in a significantly reduced cytotoxicity. Instead, more potent coibamide A derivatives with a ß-(4-biphenylyl)alanine (Bph) group were identified after the optimization of the Tyr(Me) position in the original macrocyclic scaffold of coibamide A based on the characteristic apratoxin A substructures. The similar SAR between coibamide A and apratoxin A suggests that the binding site of the Tyr(Me) side chain at the luminal end of Sec61α may be shared.

Ion identity molecular networking for mass spectrometry-based metabolomics in the GNPS environment.

Molecular networking connects mass spectra of molecules based on the similarity of their fragmentation patterns. However, during ionization, molecules commonly form multiple ion species with different fragmentation behavior. As a result, the fragmentation spectra of these ion species often remain unconnected in tandem mass spectrometry-based molecular networks, leading to redundant and disconnected sub-networks of the same compound classes. To overcome this bottleneck, we develop Ion Identity Molecular Networking (IIMN) that integrates chromatographic peak shape correlation analysis into molecular networks to connect and collapse different ion species of the same molecule. The new feature relationships improve network connectivity for structurally related molecules, can be used to reveal unknown ion-ligand complexes, enhance annotation within molecular networks, and facilitate the expansion of spectral reference libraries. IIMN is integrated into various open source feature finding tools and the GNPS environment. Moreover, IIMN-based spectral libraries with a broad coverage of ion species are publicly available.

Targeting of HER/ErbB family proteins using broad spectrum Sec61 inhibitors coibamide A and apratoxin A.

Coibamide A is a potent cancer cell toxin and one of a select group of natural products that inhibit protein entry into the secretory pathway via a direct inhibition of the Sec61 protein translocon. Many Sec61 client proteins are clinically relevant drug targets once trafficked to their final destination in or outside the cell, however the use of Sec61 inhibitors to block early biosynthesis of specific proteins is at a pre-clinical stage. In the present study we evaluated the action of coibamide A against human epidermal growth factor receptor (HER, ErbB) proteins in representative breast and lung cancer cell types. HERs were selected for this study as they represent a family of Sec61 clients that is frequently dysregulated in human cancers, including coibamide-sensitive cell types. Although coibamide A inhibits biogenesis of a broad range of Sec61 substrate proteins in a presumed substrate-nonselective manner, endogenous HER3 (ErbB-3) and EGFR (ErbB-1) proteins were more sensitive to coibamide A, and the related Sec61 inhibitor apratoxin A, than HER2 (ErbB-2). Despite this rank order of sensitivity (HER3 > EGFR > HER2), Sec61-dependent inhibition by coibamide A was sufficient to decrease cell surface expression of HER2. We report that coibamide A- or apratoxin A-mediated block of HER3 entry into the secretory pathway is unlikely to be mediated by the HER3 signal peptide alone. HER3 (G11L/S15L), that is fully resistant to the highly substrate-selective cotransin analogue CT8, was more resistant than wild-type HER3 but only at low coibamide A (3 nM) concentrations; HER3 (G11L/S15L) expression was inhibited by higher concentrations of either natural product. Time- and concentration-dependent decreases in HER protein expression induced a commensurate reduction in AKT/MAPK signaling in breast and lung cancer cell types and loss in cell viability. Coibamide A potentiated the cytotoxic efficacy of small molecule kinase inhibitors lapatinib and erlotinib in breast and lung cancer cell types, respectively. These data indicate that natural product modulators of Sec61 function have value as chemical probes to interrogate HER/ErbB signaling in treatment-resistant human cancers.

Draft Genome Sequence of Streptomyces sp. Strain ventii, Isolated from a Microbial Mat near Hydrothermal Vents within the Axial Seamount in the Pacific Ocean, and Resequencing of the Type Strains Streptomyces lonarensis NCL 716 and Streptomyces bohaiensis 11A07.

The draft genome of Streptomyces sp. strain ventii, an environmental isolate recovered from deep-sea hydrothermal vents in the Pacific Ocean, is presented along with the resequenced draft genomes of the type strains Streptomyces bohaiensis 11A07 and Streptomyces lonarensis NCL 716.

ReDU: a framework to find and reanalyze public mass spectrometry data.

We present ReDU ( https://redu.ucsd.edu/ ), a system for metadata capture of public mass spectrometry-based metabolomics data, with validated controlled vocabularies. Systematic capture of knowledge enables the reanalysis of public data and/or co-analysis of one's own data. ReDU enables multiple types of analyses, including finding chemicals and associated metadata, comparing the shared and different chemicals between groups of samples, and metadata-filtered, repository-scale molecular networking.

Coibamide A Targets Sec61 to Prevent Biogenesis of Secretory and Membrane Proteins.

Coibamide A (CbA) is a marine natural product with potent antiproliferative activity against human cancer cells and a unique selectivity profile. Despite promising antitumor activity, the mechanism of cytotoxicity and specific cellular target of CbA remain unknown. Here, we develop an optimized synthetic CbA photoaffinity probe (photo-CbA) and use it to demonstrate that CbA directly targets the Sec61α subunit of the Sec61 protein translocon. CbA binding to Sec61 results in broad substrate-nonselective inhibition of ER protein import and potent cytotoxicity against specific cancer cell lines. CbA targets a lumenal cavity of Sec61 that is partially shared with known Sec61 inhibitors, yet profiling against resistance conferring Sec61α mutations identified from human HCT116 cells suggests a distinct binding mode for CbA. Specifically, despite conferring strong resistance to all previously known Sec61 inhibitors, the Sec61α mutant R66I remains sensitive to CbA. A further unbiased screen for Sec61α resistance mutations identified the CbA-resistant mutation S71P, which confirms nonidentical binding sites for CbA and apratoxin A and supports the susceptibility of the Sec61 plug region for channel inhibition. Remarkably, CbA, apratoxin A, and ipomoeassin F do not display comparable patterns of potency and selectivity in the NCI60 panel of human cancer cell lines. Our work connecting CbA activity with selective prevention of secretory and membrane protein biogenesis by inhibition of Sec61 opens up possibilities for developing new Sec61 inhibitors with improved drug-like properties that are based on the coibamide pharmacophore.

Reproducible molecular networking of untargeted mass spectrometry data using GNPS.

Global Natural Product Social Molecular Networking (GNPS) is an interactive online small molecule-focused tandem mass spectrometry (MS2) data curation and analysis infrastructure. It is intended to provide as much chemical insight as possible into an untargeted MS2 dataset and to connect this chemical insight to the user's underlying biological questions. This can be performed within one liquid chromatography (LC)-MS2 experiment or at the repository scale. GNPS-MassIVE is a public data repository for untargeted MS2 data with sample information (metadata) and annotated MS2 spectra. These publicly accessible data can be annotated and updated with the GNPS infrastructure keeping a continuous record of all changes. This knowledge is disseminated across all public data; it is a living dataset. Molecular networking-one of the main analysis tools used within the GNPS platform-creates a structured data table that reflects the molecular diversity captured in tandem mass spectrometry experiments by computing the relationships of the MS2 spectra as spectral similarity. This protocol provides step-by-step instructions for creating reproducible, high-quality molecular networks. For training purposes, the reader is led through a 90- to 120-min procedure that starts by recalling an example public dataset and its sample information and proceeds to creating and interpreting a molecular network. Each data analysis job can be shared or cloned to disseminate the knowledge gained, thus propagating information that can lead to the discovery of molecules, metabolic pathways, and ecosystem/community interactions.

Molecular Networking Reveals Two Distinct Chemotypes in Pyrroloiminoquinone-Producing Tsitsikamma favus Sponges.

The temperate marine sponge, Tsitsikamma favus, produces pyrroloiminoquinone alkaloids with potential as anticancer drug leads. We profiled the secondary metabolite reservoir of T. favus sponges using HR-ESI-LC-MS/MS-based molecular networking analysis followed by preparative purification efforts to map the diversity of new and known pyrroloiminoquinones and related compounds in extracts of seven specimens. Molecular taxonomic identification confirmed all sponges as T. favus and five specimens (chemotype I) were found to produce mainly discorhabdins and tsitsikammamines. Remarkably, however, two specimens (chemotype II) exhibited distinct morphological and chemical characteristics: the absence of discorhabdins, only trace levels of tsitsikammamines and, instead, an abundance of unbranched and halogenated makaluvamines. Targeted chromatographic isolation provided the new makaluvamine Q, the known makaluvamines A and I, tsitsikammamine B, 14-bromo-7,8-dehydro-3-dihydro-discorhabdin C, and the related pyrrolo-ortho-quinones makaluvamine O and makaluvone. Purified compounds displayed different activity profiles in assays for topoisomerase I inhibition, DNA intercalation and antimetabolic activity against human cell lines. This is the first report of makaluvamines from a Tsitsikamma sponge species, and the first description of distinct chemotypes within a species of the Latrunculiidae family. This study sheds new light on the putative pyrroloiminoquinone biosynthetic pathway of latrunculid sponges.

Correction: The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research.

Correction for 'The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research' by James B. McAlpine et al., Nat. Prod. Rep., 2018, DOI: .

The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research.

Covering: up to 2018With contributions from the global natural product (NP) research community, and continuing the Raw Data Initiative, this review collects a comprehensive demonstration of the immense scientific value of disseminating raw nuclear magnetic resonance (NMR) data, independently of, and in parallel with, classical publishing outlets. A comprehensive compilation of historic to present-day cases as well as contemporary and future applications show that addressing the urgent need for a repository of publicly accessible raw NMR data has the potential to transform natural products (NPs) and associated fields of chemical and biomedical research. The call for advancing open sharing mechanisms for raw data is intended to enhance the transparency of experimental protocols, augment the reproducibility of reported outcomes, including biological studies, become a regular component of responsible research, and thereby enrich the integrity of NP research and related fields.

Jizanpeptins, Cyanobacterial Protease Inhibitors from a Symploca sp. Cyanobacterium Collected in the Red Sea.

Jizanpeptins A-E (1-5) are micropeptin depsipeptides isolated from a Red Sea specimen of a Symploca sp. cyanobacterium. The planar structures of the jizanpeptins were established using NMR spectroscopy and mass spectrometry and contain 3-amino-6-hydroxy-2-piperidone (Ahp) as one of eight residues in a typical micropeptin motif, as well as a side chain terminal glyceric acid sulfate moiety. The absolute configurations of the jizanpeptins were assigned using a combination of Marfey's methodology and chiral-phase HPLC analysis of hydrolysis products compared to commercial and synthesized standards. Jizanpeptins A-E showed specific inhibition of the serine protease trypsin (IC50 = 72 nM to 1 µM) compared to chymotrypsin (IC50 = 1.4 to >10 µM) in vitro and were not overtly cytotoxic to HeLa cervical or NCI-H460 lung cancer cell lines at micromolar concentrations.