Marine Sponge Theonella Swinhoei Research Articles

Onnamide A suppresses the severe acute respiratory syndrome-coronavirus 2 infection without inhibiting 3-chymotrypsin-like cysteine protease.

Given the continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of new inhibitors is necessary to enhance clinical efficacy and increase the options for combination therapy for the coronavirus disease 2019. Because marine organisms have been a resource for the discovery of numerous bioactive molecules, we constructed an extract library of marine invertebrates collected from the Okinawa Islands. In this study, the extracts were used to identify antiviral molecules against SARS-CoV-2. Using a cytopathic effect (CPE) assay in VeroE6/TMPRSS2 cells, an extract from the marine sponge Theonella swinhoei was found to reduce virus-induced CPE. Eventually, onnamide A was identified as an antiviral compound in the extract using column chromatography and NMR analysis. Onnamide A inhibited several SARS-CoV-2 variant-induced CPEs in VeroE6/TMPRSS2 cells as well as virus production in the supernatant of infected cells. Moreover, this compound blocked the entry of SARS-CoV-2 pseudo-virions. Taken together, these results demonstrate that onnamide A suppresses SARS-CoV-2 infection, which may be partially related to entry inhibition, and is expected to be a candidate lead compound for the development of anti-SARS-CoV-2 drugs.

Paenibacillus spongiae sp. nov. isolated from deep-water marine sponge Theonella swinhoei.

A novel bacterial strain, designated as PHS-Z3T, was isolated from a marine sponge belonging to the genus Theonella on the Puerto Galera Deep Monkey, Philippines. Cells of PHS-Z3T were Gram-stain-positive, motile, oxidase- and catalase-positive, white-pigmented, spore-forming, short rods that could grow at 10-40 °C (optimum, 20 °C), pH 6.0-9.5 (optimum, pH 7.5) and with 2-16 % (w/v) NaCl (optimum, 7 %). The 16S rRNA gene sequence of PHS-Z3T showed 97.9 %, 96.7 %, and 96.2 % identities to Paenibacillus mendelii C/2T, Paenibacillus oenotherae DT7-4T and Paenibacillus aurantiacus RC11T, respectively. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that PHS-Z3T formed an independent cluster with Paenibacillus mendelii C/2T. The total genome of PHS-Z3T was approximately 7 613 364 bp in size with a DNA G+C content of 51.6 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between PHS-Z3T and other type strains of species of the genus Paenibacillus were 68.0-81.4 % [ANI by blast (ANIb)], 83.0-88.0 % [ANI by MUMmer (ANIm)] and 12.7-32.1 % (dDDH). The dDDH and ANI values were below the standard cut-off criteria for delineation of bacterial species. The percentage of conserved proteins (POCP) values between the genome of PHS-Z3T and those of members of the genus Paenibacillus were 39.7-75.7 %, while the average amino acid identity (AAI) values were 55.9-83.7 %. The sole respiratory quinone in the strain was MK-7, and the predominant fatty acids were anteiso-C15 : 0 and C16 : 0. The major polar lipids of PHS-Z3T consisted of diphosphatidylglycerol, phospholipid and phosphatidylglycerol. The characteristic amino acid in the cell wall of PHS-Z3T was diamino heptanoic acid (meso-DAP). On the basis of the molecular, physiological, biochemical and chemotaxonomic features, strain PHS-Z3T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus spongiae sp. nov. is proposed, with the type strain PHS-Z3T (=MCCC 1K07848T=KCTC 43443T).

Diversity and functional roles of the symbiotic microbiome associated to marine sponges off Karah Island, Terengganu, Malaysia

Marine sponges harbor a complex microbial consortium that plays important roles in host metabolisms and the production of chemical compounds. Symbiotic microbial community structures of marine sponges have been studied in recent years especially in the Caribbean and the Mediterranean Sea, whereas there is still a paucity of research in Malaysia. This study investigated the microbial community structures associated with four different marine sponge species (Aaptos suberitoides (Brøndsted, 1934), Neopetrosia exigua (Kirkpatrick, 1900), Theonella swinhoei (Gray, 1868), and Xestospongia testudinaria (Lamarck, 1815)) collected from Karah Island, Peninsular Malaysia. Microbial genomic DNA of four sponges and surrounding seawater were extracted and 16S rRNA gene amplicon sequencing was conducted targeting V3–V4 variable region using Illumina MiSeq platform. Paired-end sequences were analyzed using QIIME2 and the potential microbial functions related to the microbial communities of marine sponges and seawater were annotated using functional annotation of prokaryotic taxa (FAPROTAX). Microbial community structures and compositions were clearly different between sponges and seawater, as well as within different sponge species. Taxonomic analysis showed that Chloroflexi was the predominant phylum in all sponge species. Proteobacteria and Actinobacteriota were composing the core taxa of the microbial communities in N. exigua, T. swinhoei, and X. testudinaria, whereas Cyanobacteria and Proteobacteria were dominant in A. suberitoides. The functional analysis revealed that the abundance of the “predatory or exoparasitic” function was high in all sponges. The functions of aerobic ammonia oxidation and nitrification were dominant in marine sponges, rather than seawater. This study has important implications in understanding the microbial community structures associated with marine sponges in Malaysia, and gives fundamental information of sponge–microbe interactions and functional roles for future investigations.

New Theonellapeptolides from Indonesian Marine Sponge Theonella swinhoei as Anti-Austerity Agents

We reported three new members of the theonellapeptolide family from theonellapeptolide II series, namely theonellapeptolides IIb (1), IIa (2), IIc (3), and three known members-IId (4), IIe (5), and Id (6)-from Kodingarengan marine sponge Theonella swinhoei collected in Makassar, Indonesia. The structures of tridecadepsipeptides 1-3, including the absolute configurations of their amino acids, were determined by the integrated NMR and tandem MS analyses followed by Marfey's analysis. To the best of our knowledge, 1 and 2 are the first theonellapeptolide-type compounds to have a valine residue with D configuration at residue position 6. The isolated theonellapeptolide-type compounds 1-6 showed selective cytotoxic activity against human pancreatic MIA PaCa-2 cancer cells in a nutrient-deprived medium. Among them, the most potent preferential cytotoxicity was observed in new theonellapeptolide IIc (3) and known IId (4), IIe (5), and Id (6).

Arsenate reducing bacteria isolated from the marine sponge Theonella swinhoei: Bioremediation potential

Arsenic (As) contamination of freshwater resources constitutes a major environmental issue affecting over 200 million people worldwide. Although the use of microorganisms for the bioremediation of As has been well studied, only very few candidates have been identified to date. Here, we investigated bacteria associated with the Red Sea sponge Theonella swinhoei and their potential to reduce As in a low-salinity liquid medium. This Indo-Pacific common sponge has been shown to hyper-accumulate As, at an average concentration of 8600 mg/g-1 in an environment uncontaminated by arsenic or barium. Four isolated strains of bacteria exhibited arsenic reduction potential by transforming inorganic As in the form of arsenate (iAsV) to arsenite (iAsIII). Two of these isolates were identified as Alteromonas macleodii and Pseudovibrio ascidisceicola, and the other two isolates, both belonging to the same species, were identified as Pseudovibrio denitrificans. The four isolates were then cultured in a low-salinity iAsV-rich medium (5 mM) and As concentration was measured over time using a specifically designed high-performance liquid chromatograph coupled to a mass spectrometer (HPLC-MS). Out of the four isolates, A. macleodii and P. ascidisceicola grew successfully in a low-salinity liquid medium and reduced AsV to AsIII at an average rate of 0.094 and 0.083 mM/h, respectively, thereby demonstrating great potential for the bioremediation of As-contaminated groundwater.

カイメンTheonella swinhoei由来新規細胞毒性ペプチドnazumazoles A-Fの単離と構造決定

カイメンTheonella swinhoei由来新規細胞毒性ペプチドnazumazoles A-Fの単離と構造決定

New 4-methylidene sterols from the marine sponge Theonella swinhoei

A series of 4-methylidene sterols including three new compounds 1–3, were isolated from the marine sponge Theonella swinhoei. The structures of new compounds were determined on the basis of spectroscopic analyses. Compounds 3, 5, and 6 showed cytotoxicities against U937, MCF-7, and PC-9 cancer cells with IC50 in the range of 1.6–8.8 μM. The new compound 3 exhibited remarkable proapoptotic activity in breast cancer cells. Mechanically, 3 significantly triggered reactive oxygen species (ROS) accumulation resulting in apoptosis and DNA damage in breast cancer cells.

Metagenomics-Based Cloning of Amilase-Encoding Genes from the Uncultured Symbiotic Bacteria of a Marine Sponge Theonella swinhoei from Kapoposang Island, South Sulawesi

Marine sponges have recently been recognized as the source ofenzymes, including members of hydrolases. Hydrolytic enzymes are extracellularly produced by sponge-associated bacteria to mediate the metabolism of complex organic matters, thereby assisting the sponge hosts in nutrition and metabolic processes. Among hydrolytic enzymes, amilaseshas attracted increasing attention due to their potential industrial applications. This research work was aimed atutilizing functional metagenomicsapproach for the discovery of amilases derived from the uncultured symbiotic bacteriaof the Indonesian marine sponge Theonella swinhoei. Weinitially constructed a small-insert metagenomiclibrary in Escherichia coliby cloning of metagenome in the size range of5-20 kb prepared from the sponge’s microbiome. Further functional screening of the resulting metagenomic library led to the isolation of two recombinant E. coli clones potentially harboring amilase genes, as indicated by the presence of clearing zones surroinding the selective medium containing 1% amilum.

Total Synthesis of Theonellapeptolide Id.

Theonellapeptolide Id is a tridecapeptide containing a 37-membered lactone, originally isolated from the marine sponge Theonella swinhoei. In addition to moderate cytotoxicity, immunosuppressive activity had been reported for this natural cyclodepsipeptide. However, the synthetic material to verify its unique biological activity has not been available thus far. In this study, the first total synthesis of theonellapeptolide Id has been performed by solid phase peptide synthesis, and the biological activity has been confirmed in comparison with cyclosporin A.

Insights into the lifestyle of uncultured bacterial natural product factories associated with marine sponges

The as-yet uncultured filamentous bacteria "Candidatus Entotheonella factor" and "Candidatus Entotheonella gemina" live associated with the marine sponge Theonella swinhoei Y, the source of numerous unusual bioactive natural products. Belonging to the proposed candidate phylum "Tectomicrobia," Candidatus Entotheonella members are only distantly related to any cultivated organism. The Ca E. factor has been identified as the source of almost all polyketide and modified peptides families reported from the sponge host, and both Ca Entotheonella phylotypes contain numerous additional genes for as-yet unknown metabolites. Here, we provide insights into the biology of these remarkable bacteria using genomic, (meta)proteomic, and chemical methods. The data suggest a metabolic model of Ca Entotheonella as facultative anaerobic, organotrophic organisms with the ability to use methanol as an energy source. The symbionts appear to be auxotrophic for some vitamins, but have the potential to produce most amino acids as well as rare cofactors like coenzyme F420 The latter likely accounts for the strong autofluorescence of Ca Entotheonella filaments. A large expansion of protein families involved in regulation and conversion of organic molecules indicates roles in host-bacterial interaction. In addition, a massive overrepresentation of members of the luciferase-like monooxygenase superfamily points toward an important role of these proteins in Ca Entotheonella. Furthermore, we performed mass spectrometric imaging combined with fluorescence in situ hybridization to localize Ca Entotheonella and some of the bioactive natural products in the sponge tissue. These metabolic insights into a new candidate phylum offer hints on the targeted cultivation of the chemically most prolific microorganisms known from microbial dark matter.

The importance of N-methylations for the stability of the β⁶·³-helical conformation of polytheonamide B.

Polytheonamide B (pTB), a highly cytotoxic peptide produced by a symbiotic bacterium of the marine sponge Theonella swinhoei, forms a transmembrane pore consisting of 49 residues. More than half of its residues are posttranslationally modified. Epimerizations result in alternating L- and D-amino acids that allow the peptide to adopt a [Formula: see text]-helical conformation. Unusually, the wide [Formula: see text]-helix of pTB is stable in a polar environment, which is in contrast to gramicidin A, an antibiotic with similar function and structure. The role of the other posttranslational modifications (PTMs) such as side chain hydroxylations, C- and N-methylations is not well understood. In this study, the importance of these PTMs for the stability of [Formula: see text]-helix is investigated using computational tools. By reverting the modified residues to their precursors and monitoring the effect on the dominant structure, we show that the N-methylations are crucial for the stability of the [Formula: see text]-helix in a polar environment. They are the driving force for the formation of stable side chain hydrogen-bond chains that act as an "exoskeleton." Such exoskeletons could present a general design strategy for helical peptides.

Yakushinamides, Polyoxygenated Fatty Acid Amides That Inhibit HDACs and SIRTs, from the Marine Sponge Theonella swinhoei.

Yakushinamides A (1) and B (2), prolyl amides of polyoxygenated fatty acids, have been isolated from the marine sponge Theonella swinhoei as inhibitors of HDACs and SIRTs. Their planar structures were determined by interpretation of the NMR data of the intact molecules and tandem FABMS data of the methanolysis products. For the assignment of the relative configurations of the three contiguous oxymethine carbons in 1 and 2, Kishi's universal NMR database was applied to the methanolysis products. During the assignments of relative configurations of the isolated 1-hydroxy-3-methyl moiety in 1 and the isolated 1-hydroxy-2-methyl moiety in 2, we found diagnostic NMR features to distinguish each pair of diastereomers. The absolute configurations of 1 and 2 were determined by a combination of the modified Mosher's method and Marfey's method. Although the modified Mosher's method was successfully applied to the methanolysis product of 1, this method gave an ambiguous result at C-20 when applied to the methanolysis product of 2, even after oxidative cleavage of the C-14 and C-15 bond.

Total Synthesis of Aurantoside G, an N-β-Glycosylated 3-Oligoenoyltetramic Acid from Theonella swinhoei.

The first synthesis of a natural N-glycosylated 3-acyltetramic acid is reported. Aurantoside G (1 g), a deep-red metabolite of the marine sponge Theonella swinhoei, is highly delicate in the pure state. It features a chlorinated dodecapentaenoyl side chain at an l-asparagine-derived tetramic acid, the ring nitrogen atom of which is linked to a β-configured d-xylose. The side chain was built through consecutive Wittig and HWE reactions and used to N-acylate the amino group of an asparaginate that had already been N-xylosylated through a Fukuyama-Mitsunobu reaction. This N-acylation step fixes the β-configuration of the xylose, which is essential for the antifungal activity, but only if the sugar carries bulky, electron-rich protecting groups such as PMB. In the final step, the heterocycle was closed quantitatively through a basic Lacey-Dieckmann condensation of an entirely unprotected precursor.

Nazumazoles D-F, Cyclic Pentapeptides That Inhibit Chymotrypsin, from the Marine Sponge Theonella swinhoei.

Nazumazoles D-F (1-3) were isolated from the marine sponge Theonella swinhoei. The compounds gave extremely broad peaks by reversed-phase HPLC using an ODS column. HPLC using a gel permeation column was instrumental for the separation of the three compounds. Their planar structures were determined by interpretation of NMR data to be cyclic pentapeptides. Nazumazoles D-F contained one residue each of α-keto-l-norvaline (l-Knv) {or α-keto-d-leucine (l-Kle)}, l-alanyloxazole (l-Aox), d-Abu (or d-Ser), N-α-CHO-β-l-Dpr, and cis-4-methyl-l-proline. The absolute configuration of each amino acid residue was determined by Marfey's method in combination with conversion of the α-keto-β-amino acid to the α-amino acid by oxidation. Nazumazoles D-F are not cytotoxic against P388 cells at 50 μM, but inhibit chymotrypsin.

Nazumazoles A-C, Cyclic Pentapeptides Dimerized through a Disulfide Bond from the Marine Sponge Theonella swinhoei.

A mixture of nazumazoles A-C (1-3) was purified from the extract of the marine sponge Theonella swinhoei. The mixture was eluted as an extraordinarily broad peak in the reversed-phase HPLC. The structures of nazumazoles were determined by interpretation of the NMR data and chemical degradations. Nazumazoles contain one residue each of alanine-derived oxazole and α-keto-β-amino acid residue. Nazumazoles exhibited cytotoxicity against P388 cells.

A talented genus

Members of a newly described candidate bacterial genus, Entotheonella, have been identified as the sources of the rich array of natural products found in the marine sponge Theonella swinhoei. Two scientists discuss this discovery from the perspectives of microbial ecology and drug discovery. See Article p.58 Almost all drugs and drug candidates from bacteria are produced by a few groups of metabolically rich organisms. That leaves the unculturable — or uncultivated — microbial majority as a largely untapped resource. Here Jorn Piel and colleagues report the use of single-cell and metagenomic analysis to identify two potential 'environmental factories', both members of the candidate genus Entotheonella and symbionts of the chemically rich marine sponge Theonella swinhoei. Importantly they find that the genomes of both microbes encode multiple distinct biosynthetic gene clusters that together account for most of the bioactive polyketides and peptides previously thought to be produced by the sponge host. This discovery identifies Entotheonella and members of the newly proposed phylum Tectomicrobia as a 'biochemically talented' phylum on a par with the actinomycetes.

An environmental bacterial taxon with a large and distinct metabolic repertoire

Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such 'talented' producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus 'Entotheonella' with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. 'Entotheonella' spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum 'Tectomicrobia'. The pronounced bioactivities and chemical uniqueness of 'Entotheonella' compounds provide significant opportunities for ecological studies and drug discovery.

Solomonsterol A, a Marine Pregnane-X-Receptor Agonist, Attenuates Inflammation and Immune Dysfunction in a Mouse Model of Arthritis

In the present study we provide evidence that solomonsterol A, a selective pregnane X receptor (PXR) agonist isolated from the marine sponge Theonella swinhoei, exerts anti-inflammatory activity and attenuates systemic inflammation and immune dysfunction in a mouse model of rheumatoid arthritis. Solomonsterol A was effective in protecting against the development of arthritis induced by injecting transgenic mice harboring a humanized PXR, with anti-collagen antibodies (CAIA) with beneficial effects on joint histopathology and local inflammatory response reducing the expression of inflammatory markers (TNFα, IFNγ and IL-17 and chemokines MIP1α and RANTES) in draining lymph nodes. Solomonsterol A rescued mice from systemic inflammation were assessed by measuring arthritis score, CRP and cytokines in the blood. In summary, the present study provides a molecular basis for the regulation of systemic local and systemic immunity by PXR agonists.

Structural insights into Estrogen Related Receptor-β modulation: 4-Methylenesterols from Theonella swinhoei sponge as the first example of marine natural antagonists

In this paper, we report the first evidence of 4-methylenesterols, isolated from the marine sponge Theonella swinhoei, as antagonists of Estrogen Related Receptors (ERRs). The interactions of 4-methylenesterols with ERRs were investigated through a multi-parametric approach involving biological assays and molecular modelling. Here the first homology model of active and inactive conformations of the Estrogen Related Receptor β (ERRβ) is also reported, benchmarked with the well known agonists gsk4716 and genistein, and the antagonists 4-hydroxytamoxifen and diethylstilbestrol. Our proposed model could contribute to the clarification of small molecule interaction mode in the ERRβ and, notably, to the rational design of new potential and selective modulators of this emerging therapeutic target.

Chemical Construction and Structural Permutation of Potent Cytotoxin Polytheonamide B: Discovery of Artificial Peptides with Distinct Functions

Polytheonamide B (1), isolated from the marine sponge Theonella swinhoei, is a posttranslationally modified ribosomal peptide (MW 5030 Da) that displays extraordinary cytotoxicity. Among its 48 amino acid residues, this peptide includes a variety D- and L-amino acids that do not occur in proteins, and the chiralities of these amino acids alternate in sequence. These structural features induce the formation of a stable β6.3-helix, giving rise to a tubular structure of over 4 nm in length. In the biological setting, this fold is believed to transport cations across the lipid bilayer through a pore, thereby acting as an ion channel. In this Account, we discuss the construction and structural permutations of this potent cytotoxin. First we describe the 161-step chemical construction of this unusual peptide 1. By developing a synthetic route to 1, we established the chemical basis for subsequent SAR studies to pinpoint the proteinogenic and nonproteinogenic building blocks within the molecule that confer its toxicity and channel function. Using fully synthetic 1, we generated seven analogues with point mutations, and studies of their activity revealed the importance of the N-terminal moiety. Next, we simplified the structure of 1 by substituting six amino acid residues of 1 to design a more synthetically accessible analogue 9. This dansylated polytheonamide mimic 9 was synthesized in 127 total steps, and we evaluated its function to show that it can emulate the toxic and ion channel activities of 1 despite its multiple structural modifications. Finally, we applied a highly automated synthetic route to 48-mer 9 to generate 13 substructures of 27-39-mers. The 37-mer 12 exhibited nanomolar level toxicity through a potentially distinct mode of action from 1 and 9. The SAR studies of polytheonamide B and the 21 artificial analogues have deepened our understanding of the precise structural requirements for the biological functions of 1. They have also led to the discovery of artificial molecules with various toxicities and channel functions. These achievements demonstrate the benefits of total synthesis and the importance of efficient construction of complex molecules. The knowledge accumulated through these studies will be useful for the rational design of new tailor-made channel peptides and cytotoxic molecules with desired functions.