Chengkuizengella axinellae sp. nov., a symbiotic bacterium isolated from a marine sponge of the genus Axinella.

A Gram-stain-positive, strictly aerobic, creamy-white colored, endospore-forming and non-motile rods strain, designated as strain 2205SS18-9T, was isolated from a marine sponge, Axinella sp. collected from Seopseom Island, Republic of Korea. Optimal growth of strain 2205SS18-9T was observed at 25-30 °C, pH 6.5-7.0, and in the presence of 3.0% (w/v) NaCl. Cells were oxidase-positive and catalase-negative. Negative for nitrate reduction and indole production. Phylogenetic analyses based on the 16S rRNA gene and whole-genome sequences revealed that strain 2205SS18-9T formed a distinct phyletic lineage in the genus Chengkuizengella, and it was most closely related to Chengkuizengella marina YPA3-1-1T and Chengkuizengella sediminis J15A17T with 97.1 and 96.6% 16S rRNA gene sequence similarities, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between strain 2205SS18-9T and Chengkuizengella marina YPA3-1-1T were 79.0 and 21.6%, respectively. The genomic DNA G + C content was 34.1%. The genome harbors a number of host-adhesion and transporter genes, suggested that strain 2205SS18-9T may interact with its sponge host as a symbiont. Menaquinone-7 was the sole isoprenoid quinone and antieiso-C15:0 (28.5%), iso-C16:0 (25.8%), C16:1 ω7c alcohol (15.0%), and iso-C15:0 (11.2%) were detected as the major fatty acids. Polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, and an unidentified lipid. The cell-wall peptidoglycan contained lysine, alanine, glutamate, and aspartate. Based on these analyses, strain 2205SS18-9T represents a novel species of the genus Chengkuizengella, for which the name Chengkuizengella axinellae sp. nov. is proposed. The type strain is 2205SS18-9T (= KACC 23238T = LMG 33063T).

Axinellamine E, One New Pyrrololactam Alkaloid from the South China Sea Sponge Axinella sp.

Chemical investigation of the EtOH extract of the sponge Axinella sp. collected from the South China Sea resulted in the identification of one new pyrrololactam alkaloid, axinellamine E (2), along with four known analogs (1, 3-5). Compound 1 was initially separated as enantiomers and was further separated to be optically pure compounds (1 a and 1 b) by a chiral column. The planar structure of compound 2 was determined mainly by 1D-, 2D-NMR, and HR-ESI-MS data analyses. Absolute configurations of 1 a and 1 b was defined by calculated ECD spectra method. All of the compounds were evaluated for their anti-inflammatory activities against nitric oxide production in lipopolysaccharide-induced RAW 264.7 cells among which compound 1 showed weak activity at 40 µg/mL. Plausible biosynthetic pathways corresponding to aldisine analogs of 1, 2, 4, and 5 were also discussed.

Integrating Molecular Networking and 1H NMR Spectroscopy for Isolation of Bioactive Metabolites from the Persian Gulf Sponge Axinella sinoxea.

The geographic position, highly fluctuating sea temperatures and hypersalinity make Persian Gulf an extreme environment. Although this unique environment has high biodiversity dominated by invertebrates, its potential in marine biodiscovery has largely remained untapped. Herein, we aimed at a detailed analysis of the metabolome and bioactivity profiles of the marine sponge Axinella sinoxea collected from the northeast coast of the Persian Gulf in Iran. The crude extract and its Kupchan subextracts were tested in multiple in-house bioassays, and the crude extract and its CHCl3-soluble portion showed in vitro antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium (Efm). A molecular networking (MN)-based dereplication strategy by UPLC-MS/MS revealed the presence of phospholipids and steroids, while 1H NMR spectroscopy indicated the presence of additional metabolites, such as diketopiperazines (DKPs). Integrated MN and 1H NMR analyses on both the crude and CHCl3 extracts combined with an antibacterial activity-guided isolation approach afforded eight metabolites: a new diketopiperazine, (-)-cyclo(L-trans-Hyp-L-Ile) (8); a known diketopiperazine, cyclo(L-trans-Hyp-L-Phe) (7); two known phospholipids, 1-O-hexadecyl-sn-glycero-3-phosphocholine (1) and 1-O-octadecanoyl-sn-glycero-3-phosphocholine (2); two known steroids, 3ß-hydroxycholest-5-ene-7,24-dione (3) and (22E)-3ß-hydroxycholesta-5,22-diene-7,24-dione (4); two known monoterpenes, loliolide (5) and 5-epi-loliolide (6). The chemical structures of the isolates were elucidated by a combination of NMR spectroscopy, HRMS and [α]D analyses. All compounds were tested against MRSA and Efm, and compound 3 showed moderate antibacterial activity against MRSA (IC50 value 70 µg/mL). This is the first study that has dealt with chemical and bioactivity profiling of A. sinoxea leading to isolation and characterization of pure sponge metabolites.

Marine Fungus Aspergillus chevalieri TM2-S6 Extract Protects Skin Fibroblasts from Oxidative Stress.

The strain Aspergillus chevalieri TM2-S6 was isolated from the sponge Axinella and identified according to internal transcribed spacer (ITS) molecular sequence homology with Aspergillus species from the section Restricti. The strain was cultivated 9 days on potato dextrose broth (PDB), and the medium evaluated as antioxidant on primary normal human dermal fibroblasts (NHDF). The cultivation broth was submitted to sterile filtration, lyophilized and used without any further processing to give the Aspergillus chevalieri TM2-S6 cultivation broth ingredient named ACBB. ACCB contains two main compounds: tetrahydroauroglaucin and flavoglaucin. Under oxidative stress, ACCB showed a significant promotion of cell viability. To elucidate the mechanism of action, the impact on a panel of hundreds of genes involved in fibroblast physiology was evaluated. Thus, ACCB stimulates cell proliferation (VEGFA, TGFB3), antioxidant response (GPX1, SOD1, NRF2), and extracellular matrix organization (COL1A1, COL3A1, CD44, MMP14). ACCD also reduced aging (SIRT1, SIRT2, FOXO3). These findings indicate that Aspergillus chevalieri TM2-S6 cultivation broth exhibits significant in vitro skin protection of human fibroblasts under oxidative stress, making it a potential cosmetic ingredient.

Collagen from the Marine Sponges Axinella cannabina and Suberites carnosus: Isolation and Morphological, Biochemical, and Biophysical Characterization.

In search of alternative and safer sources of collagen for biomedical applications, the marine demosponges Axinella cannabina and Suberites carnosus, collected from the Aegean and the Ionian Seas, respectively, were comparatively studied for their insoluble collagen, intercellular collagen, and spongin-like collagen content. The isolated collagenous materials were morphologically, physicochemically, and biophysically characterized. Using scanning electron microscopy and transmission electron microscopy the fibrous morphology of the isolated collagens was confirmed, whereas the amino acid analysis, in conjunction with infrared spectroscopy studies, verified the characteristic for the collagen amino acid profile and its secondary structure. Furthermore, the isoelectric point and thermal behavior were determined by titration and differential scanning calorimetry, in combination with circular dichroism spectroscopic studies, respectively.

Cytotoxic Guanidine Alkaloids from a French Polynesian Monanchora n. sp. Sponge.

Four bicyclic and three pentacyclic guanidine alkaloids (1-7) were isolated from a French Polynesian Monanchora n. sp. sponge, along with the known alkaloids monalidine A (8), enantiomers 9-11 of known natural product crambescins, and the known crambescidins 12-15. Structures were assigned by spectroscopic data interpretation. The relative and absolute configurations of the alkaloids were established by analysis of (1)H NMR and NOESY spectra and by circular dichroism analysis. The new norcrambescidic acid (7) corresponds to interesting biosynthetic variation within the pentacyclic core. All compounds exhibited antiproliferative and cytotoxic efficacy against KB, HCT116, HL60, MRC5, and B16F10 cancer cells, with IC50 values ranging from 4 nM to 10 µM.

Diversity and antibacterial activity of bacteria cultured from Mediterranean Axinella spp. sponges.

AIMS: Evaluation of the diversity and antibacterial activity of bacteria cultivated from Mediterranean Axinella sponges and investigating the influence of culture conditions on antibacterial activity profiles of sponge bacteria. METHODS AND RESULTS: Based on 16S rRNA gene sequence analysis, the 259 bacteria isolated from the three Mediterranean Axinella sponges A. cannabina, A. verrucosa and A. polypoides belonged to 41 genera from the four phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria and included five potential newly cultured genera. In antagonistic streak assays, 87 isolates (34%) from 13 genera showed antibacterial activity towards at least one of the 10 environmental and laboratory test bacteria. The extracts and filtrates of 22 isolates grown under three different culture conditions were less often active as the isolates in the corresponding antagonistic streak assays. Changes in antibacterial activity profiles were isolate- and culture condition-specific. CONCLUSIONS: Axinella sponges are a good source to cultivate phylogenetic diverse and hitherto novel bacteria, many of which with antibacterial activity. Analysis of induced antibacterial activities might enhance the role of sponge bacteria in efforts to isolate new antibiotics in the future. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to investigate the diversity and antibacterial activity of bacteria isolated from A. cannabina and A. verrucosa. It highlights the potential importance of induced activity and the need for employing multiple culture conditions in antibacterial screening assays of sponge-associated bacteria.

Luteivirga sdotyamensis gen. nov., sp. nov., a novel bacterium of the phylum Bacteroidetes isolated from the Mediterranean sponge Axinella polypoides.

A novel aerobic bacterium, designated strain PIII.02(T), was isolated from a Mediterranean sponge (Axinella polypoides) collected off the Israeli coast near Sdot Yam. The non-motile cells were Gram-staining-negative, oxidase-positive and catalase-positive. The orange pigment of colonies growing on marine agar was neither diffusible nor flexirubin-like. Strain PIII.02(T) grew at 15-35 °C, at pH 6.0-9.0, with 2.0-7.0 % (w/v) NaCl, and with 1.0-8.0 % (w/v) sea salts. The predominant fatty acids were iso-C15 : 0, iso-C16 : 1 H, iso-C16 : 0, C16 : 0, anteiso-C15 : 0 and C16 : 1ω7c. The major respiratory quinone was MK-7. The genomic DNA G+C content of the novel strain was 38.1 mol%. Results from 16S rRNA gene sequence analysis indicated that strain PIII.02(T) was distantly related to established members of the phylum Bacteroidetes. The established species found to be most closely related to the novel strain was Persicobacter diffluens NCIMB 1402(T) (87.6 % 16S rRNA gene sequence similarity). Based on the phenotypic and chemotaxonomic data and the results of the phylogenetic analyses, strain PIII.02(T) represents a novel species of a new genus, for which the name Luteivirga sdotyamensis gen. nov., sp. nov. is proposed. The type strain is PIII.02(T) ( = ATCC BAA-2393(T)  = LMG 26723(T)).

Aureivirga marina gen. nov., sp. nov., a marine bacterium isolated from the Mediterranean sponge Axinella verrucosa.

Two bacterial strains, VI.14 and VIII.04(T), were isolated from the Mediterranean sponge Axinella verrucosa collected off the Israeli coast near Sdot Yam. The non-motile, aerobic, Gram-negative isolates were oxidase-negative and catalase-positive, and formed golden-brown colonies on marine agar 2216. The pigment was neither diffusible nor flexirubin-like. Strain VIII.04(T) grew at 15-37 °C, at pH 6.0-9.0, in the presence of 20-50 g NaCl l(-1) and 20-80 g sea salts l(-1), The spectrum was narrower for strain VI.14, with growth at pH 7.0-8.0. and in the presence of 30-50 g NaCl l(-1) and 30-70 g sea salts l(-1). The predominant fatty acid (>50 %) in both strains was iso-C15 : 0, and the major respiratory quinone was MK-6. The DNA G+C content was 30.7 and 31.1 mol% for VIII.04(T) and VI.14, respectively. Results from 16S rRNA sequence similarity and phylogenetic analyses indicated that both strains are closely related to members of the family Flavobacteriaceae within the phylum Bacteroidetes, with as much as 91.7 % 16S rRNA sequence similarity. On the basis of data from the polyphasic analysis, we suggest that the strains represent a novel species in a new genus within the family Flavobacteriaceae, for which the name Aureivirga marina gen. nov., sp. nov. is proposed. Strain VIII.04(T) ( = ATCC BAA-2394(T) = LMG 26721(T)) is the type strain of Aureivirga marina.

Fulvitalea axinellae gen. nov., sp. nov., a member of the family Flammeovirgaceae isolated from the Mediterranean sponge Axinella verrucosa.

The yellow-pigmented, non-motile, Gram-negative, strictly aerobic, rod-shaped bacterial strain VI.18(T) was isolated from the Mediterranean sponge Axinella verrucosa collected off the coast near Sdot Yam, Israel. Results from 16S rRNA gene sequence analysis indicated that the isolate belonged to the family Flammeovirgaceae. The highest nucleotide similarity (91.4 %) occurred with Aureibacter tunicatorum A5Q-118(T). The predominant cellular fatty acids of strain VI.18(T) were iso-C15 : 0 (56.0 %), iso-C17 : 1ω9c (22.8 %) and C16 : 0 (7.4 %) and its major respiratory quinone was MK-7. The DNA G+C content was 47.5 mol%. The strain could readily be distinguished from its phylogenetically closest relatives by phenotypic, physiological and chemotaxonomic properties. On the basis of the data from the present polyphasic study, we propose a novel genus and species within the family Flammeovirgaceae, with the name Fulvitalea axinellae gen. nov., sp. nov. Strain VI.18(T) ( = ATCC BAA-2395(T)  = LMG 26722(T)) is the type strain of Fulvitalea axinellae.

Further investigation of the Mediterranean sponge Axinella polypoides: isolation of a new cyclonucleoside and a new betaine.

An exhaustive exploration into the metabolic content of the Mediterranean sponge Axinella-polypoides resulted in the isolation of the new betaine 5 and the new cyclonucleoside 8. The structures of the new metabolites were elucidated by spectroscopic methods assisted by computational methods. The analysis also provided evidence that the sponge does not elaborate pyrrole-imidazole alkaloids (PIAs) but, interestingly, it was shown to contain two already known cyclodipeptides, compounds 9 (verpacamide A) and 10.

Axinellasins A-D, Immunosuppressive Cycloheptapeptide Diastereomers, Discovered via a Precursor Ion Scanning-Supercritical Fluid Chromatography Strategy from the Marine Sponge Axinella species.

The precursor ion scanning-supercritical fluid chromatography (PI-SFC) method was applied to explore new methionine sulfoxide-containing cycloheptapeptides, axinellasins A-D (1-4), from the marine sponge Axinella sp. Their structures, including absolute configurations, were elucidated by detailed spectroscopic analyses and X-ray crystallography. The total synthesis of 4 was completed via an Fmoc solid/solution-phase synthesis. Compounds 1-4 exhibited immunosuppressive effects via inhibition of T and B cell proliferation, and 1 and 4 showed better inhibitory activities than their corresponding diastereomers.

New insight into marine alkaloid metabolic pathways: revisiting oroidin biosynthesis.

Sponge natural product biosynthesis: A highly sensitive in vivo protocol based on (14)C radiolabeled precursors and beta-imager autoradiography allowed the unraveling of the origin of the pyrrole 2-aminoimidazole-containing key biosynthetic intermediate oroidin. Proline and lysine are now proposed as the early precursors of the pyrrole and the 2-aminoimidazole moieties of oroidin respectively.

Anti-malarial, anti-algal, anti-tubercular, anti-bacterial, anti-photosynthetic, and anti-fouling activity of diterpene and diterpene isonitriles from the tropical marine sponge Cymbastela hooperi.

In an investigation into their potential ecological role(s), a group of mainly diterpene isonitriles, nine in total, isolated from the tropical marine sponge Cymbastela hooperi, and the sesquiterpene axisonitrile-3, isolated from the tropical marine sponge Acanthella kletra, were evaluated in a series of bioassays including anti-fouling, anti-algal, anti-photosynthetic, anti-bacterial (Gram +ve and -ve), anti-fungal, and anti-tubercular. The results of these assays showed that all of the tested compounds, with the exception of diterpene 9, were active in at least two of the applied test systems, with axisonitrile-3 (10) and diterpene isonitrile 1 being the two most active compounds overall, closely followed by diterpene isonitrile 3. Based on the results of the photosynthetic study a molecular modelling investigation was undertaken with all of the compounds used in that study. The results showed a positive correlation between reduction in photosynthetic activity and the interaction of the modelled compounds with a potential enzyme active site.

New tetromycin derivatives with anti-trypanosomal and protease inhibitory activities.

Four new tetromycin derivatives, tetromycins 1-4 and a previously known one, tetromycin B (5) were isolated from Streptomyces axinellae Pol001(T) cultivated from the Mediterranean sponge Axinella polypoides. Structures were assigned using extensive 1D and 2D NMR spectroscopy as well as HRESIMS analysis. The compounds were tested for antiparasitic activities against Leishmania major and Trypanosoma brucei, and for protease inhibition against several cysteine proteases such as falcipain, rhodesain, cathepsin L, cathepsin B, and viral proteases SARS-CoV M(pro), and PL(pro). The compounds showed antiparasitic activities against T. brucei and time-dependent inhibition of cathepsin L-like proteases with K(i) values in the low micromolar range.

Cultivation of fractionated cells from a bioactive-alkaloid-bearing marine sponge Axinella sp.

Sponges are among the most primitive multicellular organisms and well-known as a major source of marine natural products. Cultivation of sponge cells has long been an attractive topic due to the prominent evolutionary and cytological significance of sponges and as a potential approach to supply sponge-derived compounds. Sponge cell culture is carried out through culturing organized cell aggregates called 'primmorphs.' Most research culturing sponge cells has used unfractionated cells to develop primmorphs. In the current study, a tropical marine sponge Axinella sp., which contains the bioactive alkaloids, debromohymenialdisine (DBH), and hymenialdisine (HD), was used to obtain fractionated cells and the corresponding primmorphs. These alkaloids, DBH and HD, reportedly show pharmacological activities for treating osteoarthritis and Alzheimer's disease. Three different cell fractions were obtained, including enriched spherulous cells, large mesohyl cells, and small epithelial cells. These cell fractions were cultivated separately, forming aggregates that later developed into different kinds of primmorphs. The three kinds of primmorphs obtained were compared as regards to appearance, morphogenesis, and cellular composition. Additionally, the amount of alkaloid in the primmorphs-culture system was examined over a 30-d culturing period. During the culturing of enriched spherulous cells and developed primmorphs, the total amount of alkaloid declined notably. In addition, the speculation of alkaloid secretion and some phenomena that occurred during cell culturing are discussed.

Polyphyly of the genus Axinella and of the family Axinellidae (Porifera: Demospongiaep).

The genus Axinella is difficult to define on the basis of morphological characters and includes a heterogeneous assemblage of species. Several previous authors have suspected the polyphyly of both this genus and the family Axinellidae. To clarify the phylogeny of Axinellidae and Axinella, we propose a new hypothesis based on two molecular markers. In our analyses, Axinellidae and Axinella are polyphyletic assemblages. The 15 species of Axinellidae in our dataset belong to five clades and the nine species of Axinella to three clades. One Axinella clade, named Axinella(p), contains the type-species of the genus: A. polypoides (plus A. aruensis, A. dissimilis, A. infundibuliformis and A. vaceleti). A new clade, Cymbaxinella(p), is proposed, following the PhyloCode, it includes C. damicornis, C. verrucosa, C. corrugata and C. cantharella. The species Axinella cannabina is reallocated to a clade named Acanthella(p). The clades Agelas(p) and Cymbaxinella(p) constitute a new clade: Agelasida(p). Few morphological, biochemical and secondary structures characters support these groupings, highlighting the need for new characters for such problematic sponge groups. This work is an attempt to build a framework for the phylogeny of taxa allocated to Axinella and Axinellidae in the traditional classification.

Polyaxibetaine, an amino acid derivative from the marine sponge Axinella polypoides.

A new pyridinium derivative, polyaxibetaine (3), has been isolated from the marine sponge Axinella polypoides, together with two known modified amino acids, 1 and 2. The planar structure of compound 3 has been elucidated by spectroscopic methods; definition of the absolute configuration of compounds 1-3 has been carried out through ECD studies.

Bromopyrrole alkaloids as lead compounds against protozoan parasites.

In the present study, 13 bromopyrrole alkaloids, including the oroidin analogs hymenidin (2), dispacamide B (3) and dispacamide D (4), stevensine (5) and spongiacidin B (6), their derivatives lacking the imidazole ring bromoaldisin (7), longamide B (8) and longamide A (9), the dimeric oroidin derivatives sceptrin (10) and dibromopalau'amine (11), and the non-oroidin bromopyrrolohomoarginin (12), manzacidin A (13), and agelongine (14), obtained from marine sponges belonging to Axinella and Agelas genera have been screened in vitro against four parasitic protozoa, i.e., two Trypanosoma species (T. brucei rhodesiense and T. cruzi), Leishmania donovani and Plasmodium falciparum (K1 strain, a chloroquine resistant strain), responsible of human diseases with high morbidity and, in the case of malaria, high mortality. Our results indicate longamide B (8) and dibromopalau'amine (11) to be promising trypanocidal and antileishmanial agents, while dispacamide B (3) and spongiacidin B (6) emerge as antimalarial lead compounds. In addition, evaluation of the activity of the test alkaloids (2-14) against three different enzymes (PfFabI, PfFabG, PfFabZ) involved in the de novo fatty acid biosynthesis pathway of P. falciparum (PfFAS-II) identified bromopyrrolohomoarginin (12) as a potent inhibitor of PfFabZ. The structural similarity within the series of tested molecules allowed us to draw some preliminary structure-activity relationships. Tests against the mammalian L6 cells revealed important clues on therapeutic index of the metabolites. This is the first detailed study on the antiprotozoal potential of marine bromopyrrole alkaloids.

[Study on natural products for drug development].

The ubiquitin-proteasome system (UPS) plays a major role in selective protein degradation and regulates various cellular events. Approval of bortezomib for the treatment of multiple myeloma validated the proteasome as an anticancer target. In order to find drug candidates targeting the ubiquitin-dependent protein degradation, we paid an attention to inhibitors against three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3), which are required for polyubiquitination of proteins and prerequisite to proteasome-mediated protein degradation. We succeeded in isolating various compounds with three distinct inhibitory activities against an E1 enzyme reaction, Ubc13 (E2)-Uev1A interaction, and p53-HDM2 (E3) interaction as well as the proteasome inhibitors. We also isolated new alkaloids, notoamides, from a marine-derived Aspergillus sp. Among them, notoamide B and stephacidin A contain a bicyclo[2.2.2]diazaoctane ring in their structures. We proposed this ring is constructed from notoamide E by the intramolecular Diels-Alder (IMDA) reaction. Recently, the isolation of the antipodes of notoamides from the terrestrial Aspergillus has been reported. We propose that each enantiomer is generated by a distinct face-selective IMDA.