A tetramic acid derivative with protein tyrosine phosphatase 1B inhibitory activity and a new nortriterpene glycoside from the Indonesian marine sponge Petrosia sp.

During the search for protein tyrosine phosphatase 1B (PTP1B) inhibitors from marine organisms, the known tetramic acid derivative, melophlin C (1), was isolated as an active component together with the new nortriterpenoid saponin, sarasinoside S (2), and three homologues: sarasinosides A₁ (3), I₁ (4), and J (5), from the Indonesian marine sponge Petrosia sp. The structure of 2 was elucidated on the basis of its spectroscopic data. Compound 1 inhibited PTP1B activity with an IC₅₀ value of 14.6 μM, while compounds 2–5 were not active at 15.2–16.0 μM. This is the first study to report the inhibitory effects of a tetramic acid derivative on PTP1B activity.     

Puupehanol,a sesquiterpene-dihydroquinone derivative from the marine sponge Hyrtios sp.

Puupehanol (1), a new sesquiterpene-dihydroquinone derivative, was isolated from the marine sponge Hyrtios sp., along with the known compounds puupehenone (2) and chloropuupehenone (3) that are responsible for the antifungal activity observed in the extract. The structure of 1 was established as (20R,21R)-21-hydroxy-20,21-dihydropuupehenone by extensive spectroscopic and computational methods. Compound 2 exhibited potent activity against Cryptococcus neoformans and Candida krusei with MFCs of 1.25 and 2.50 μg/mL, respectively.     

The Bromotyrosine Derivative Ianthelline Isolated from the Arctic Marine Sponge Stryphnus fortis Inhibits Marine Micro- and Macrobiofouling

The inhibition of marine biofouling by the bromotyrosine derivative ianthelline, isolated from the Arctic marine sponge Stryphnus fortis, is described. All major stages of the fouling process are investigated. The effect of ianthelline on adhesion and growth of marine bacteria and microalgae is tested to investigate its influence on the initial microfouling process comparing with the known marine antifoulant barettin as a reference. Macrofouling is studied via barnacle (Balanus improvisus) settlement assays and blue mussel (Mytilus edulis) phenoloxidase inhibition. Ianthelline is shown to inhibit both marine micro- and macrofoulers with a pronounced effect on marine bacteria (minimum inhibitory concentration (MIC) values 0.1–10 μg/mL) and barnacle larval settlement (IC₅₀?=?3.0 μg/mL). Moderate effects are recorded on M. edulis (IC₅₀?=?45.2 μg/mL) and microalgae, where growth is more affected than surface adhesion. The effect of ianthelline is also investigated against human pathogenic bacteria. Ianthelline displayed low micromolar MIC values against several bacterial strains, both Gram positive and Gram negative, down to 2.5 μg/mL. In summary, the effect of ianthelline on 20 different representative marine antifouling organisms and seven human pathogenic bacterial strains is presented.     

Unusual Symbiotic Cyanobacteria Association in the Genetically Diverse Intertidal Marine Sponge Hymeniacidon perlevis (Demospongiae,Halichondrida)

Cyanobacteria represent one of the most common members of the sponge-associated bacterial community and are abundant symbionts of coral reef ecosystems. In this study we used Transmission Electron Microscopy (TEM) and molecular techniques (16S rRNA gene marker) to characterize the spatial distribution of cyanobionts in the widely dispersed marine intertidal sponge Hymeniacidon perlevis along the coast of Portugal (Atlantic Ocean). We described new sponge associated cyanobacterial morphotypes (Xenococcus-like) and we further observed Acaryochloris sp. as a sponge symbiont, previously only reported in association with ascidians. Besides these two unique cyanobacteria, H. perlevis predominantly harbored Synechococcus sp. and uncultured marine cyanobacteria. Our study supports the hypothesis that the community of sponge cyanobionts varies irrespective of the geographical location and is likely influenced by seasonal fluctuations. The observed multiple cyanobacterial association among sponges of the same host species over a large distance may be attributed to horizontal transfer of symbionts. This may explain the absence of a co-evolutionary pattern between the sponge host and its symbionts. Finally, in spite of the short geographic sampling distance covered, we observed an unexpected high intra-specific genetic diversity in H. perlevis using the mitochondrial genes ATP6 (π = 0.00177), COI (π = 0.00241) and intergenic spacer SP1 (π = 0.00277) relative to the levels of genetic variation of marine sponges elsewhere. Our study suggests that genotypic variation among the sponge host H. perlevis and the associated symbiotic cyanobacteria diversity may be larger than previously recognized.     

Optimization and production of pyrrolidone antimicrobial agent from marine sponge-associated Streptomyces sp. MAPS15

Twenty-nine actinobacterial strains were isolated from marine sponge Spongia officinalis and screened for antagonistic activity against various bacterial and fungal pathogens. The active antibiotic producer MAPS15 was identified as Streptomyces sp. using 16S rRNA phylogenetic analysis. The critical control factors were selected from Plackett–Burman (PB) factorial design and the bioprocess medium was optimized by central composite design (CCD) for the production of bioactive metabolite from Streptomyces sp. MAPS15. The maximum biomass and active compound production obtained with optimized medium was 6.13 g/L and 62.41 mg/L, respectively. The economical carbon source, paddy straw was applied for the enhanced production of bioactive compound. The purified active fraction was characterized and predicted as pyrrolidone derivative which showed broad spectrum of bioactivity towards indicator organisms. The predicted antimicrobial spectra suggested that the Streptomyces sp. MAPS15 can produce a suite of novel antimicrobial drugs.     

Isolation and diversity of planctomycetes from the sponge Niphates sp., seawater, and sediment of Moreton Bay, Australia

Planctomycetes are ubiquitous in marine environment and were reported to occur in association with multicellular eukaryotic organisms such as marine macroalgae and invertebrates. Here, we investigate planctomycetes associated with the marine sponge Niphates sp. from the sub-tropical Australian coast by assessing their diversity using culture-dependent and -independent approaches based on the 16S rRNA gene. The culture-dependent approach resulted in the isolation of a large collection of diverse planctomycetes including some novel lineages of Planctomycetes from the sponge as well as sediment and seawater of Moreton Bay where this sponge occurs. The characterization of these novel planctomycetes revealed that cells of one unique strain do not possess condensed nucleoids, a phenotype distinct from other planctomycetes. In addition, a culture-independent clone library approach identified unique planctomycete 16S rRNA gene sequences closely related to other sponge-derived sequences. The analysis of tissue of the sponge Niphates sp. showed that the mesohyl of the sponge is almost devoid of microbial cells, indicating this species is in the group of ‘low microbial abundant’ (LMA) sponges. The unique planctomycete 16S rRNA gene sequences identified in this study were phylogenetically closely related to sequences from LMA sponges in other published studies. This study has revealed new insights into the diversity of planctomycetes in the marine environment and the association of planctomycetes with marine sponges.     

Analysis of bacterial communities in sponges and coral inhabiting Red Sea,using barcoded 454 pyrosequencing

Microbial communities are linked with marine sponge are diverse in their structure and function. Our understanding of the sponge-associated microbial diversity is limited especially from Red Sea in Saudi Arabia where few species of sponges have been studied. Here we used pyrosequencing to study two marine sponges and coral species sampled from Obhur region from Red sea in Jeddah. A total of 168 operational taxonomic units (OTUs) were identified from Haliclona caerulea, Stylissa carteri and Rhytisma fulvum. Taxonomic identification of tag sequences of 16S ribosomal RNA revealed 6 different bacterial phyla and 9 different classes. A proportion of unclassified reads were was also observed in sponges and coral sample. We found diverse bacterial communities associated with two sponges and a coral sample. Diversity and richness estimates based on OUTs revealed that sponge H. caerulea had significantly high bacterial diversity. The identified OTUs showed unique clustering in three sponge samples as revealed by Principal coordinate analysis (PCoA). Proteobacteria (88–95%) was dominant phyla alonwith Bacteroidetes, Planctomycetes, Cyanobacteria, Firmicutes and Nitrospirae. Seventeen different genera were identified where genus Pseudoalteromonas was dominant in all three samples. This is first study to assess bacterial communities of sponge and coral sample that have never been studied before to unravel their microbial communities using 454-pyrosequencing method.     

Sterol composition of three marine sponge species from the genus Cinachyrella

• 1.1. The hitherto undescribed sterol compositions of three marine sponge species belonging to the genus Cinachyrella are reported: C. alloclada and C. kükenthali from the Senegalese coast, at two different depths, and C. aff. schulzei from the lagoon of Nouméa, New Caledonia.
• 2.2. Fourteen free sterols have been identified by GC and GC/MS studies, including the 23,24ξ-dimethylcholesta-5,22-dien-3β-ol (10) and the rare 24-norcholesta-5,22-dien-3β-ol (1).
• 3.3. The first compound (10) is reported for the second time in a marine sponge and it was found only in Senegalese sponges collected in shallow waters.
• 4.4. Sterol (10) has been isolated by HPLC and identified by NMR techniques.
• 5.5. Significant amounts of cholest-7-en-3β-ol (7) were also found in the Senegalese sponge species.
• 6.6. Apart from these two compounds, the three sponge sterol compositions are found to be very similar.

     

Polyacelylenic alcohols and alkaloid derivatives from a calcareous marine sponge,Leucetta chagosensis

A chemical investigation of the CH₂Cl₂ extract of the marine sponge Leucetta chagosensis afforded 8 secondary metabolites, namely, pellynol A (1), pellynol B (2), pellynol I (3), pellynol D (4), plakohypaphorine B (5), plakohypaphorine E (6), 4-bromo-1H-pyrrole-3-carboxamide (7), and 2-phenylacetamide (8). The structures of these secondary metabolites were elucidated via NMR spectroscopic analysis, MS experiment and compared with those reported in the literature. This is the first report of compounds 1–8 isolated from the marine sponge genus Leucetta and from the class Calcarea. This work contributes expands the knowledge of the chemical diversity of calcareous marine sponges, and the chemotaxonomic significance of the isolates is also discussed.     

Tetradehydrohalicyclamine B,a new proteasome inhibitor from the marine sponge Acanthostrongylophora ingens

A new halicyclamine derivative, tetradehydrohalicyclamine B (1), was isolated from the marine sponge Acanthostrongylophora ingens, along with halicyclamine B (2) as proteasome inhibitors. Compound 1 is the second example found to have a pyridinium ring in the halicyclamine family. Although the relative configuration of 2 was previously determined by X-ray crystallographic analysis, here we determined the absolute configuration of 2 by ECD experiment. Compounds 1 and 2 inhibited the constitutive proteasome as well as the immunoproteasome. The inhibitory activities of 2 were 4- to 10-fold more potent than those of 1.     

Two new antioxidant actinosporin analogues from the calcium alginate beads culture of sponge-associated Actinokineospora sp. strain EG49

Marine sponge-associated actinomycetes represent an exciting new resource for the identification of new and novel natural products . Previously, we have reported the isolation and structural elucidation of actinosporins A (1) and B (2) from Actinokineospora sp. strain EG49 isolated from the marine sponge Spheciospongia vagabunda. Herein, by employing different fermentation conditions on the same microorganism, we report on the isolation and antioxidant activity of structurally related metabolites, actinosporins C (3) and D (4). The antioxidant potential of actinosporins C and D was demonstrated using the ferric reducing antioxidant power (FRAP) assay. Additionally, at 1.25 μM, actinosporins C and D showed a significant antioxidant and protective capacity from the genomic damage induced by hydrogen peroxide in the human promyelocytic (HL-60) cell line.     

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.     

Purpuroines A–J,halogenated alkaloids from the sponge Iotrochota purpurea with antibiotic activity and regulation of tyrosine kinases

Ten new halogenated alkaloids named purpuroines A–J (1–10), and a known analogue (11), were isolated from the marine sponge Iotrochota purpurea. Their structures were elucidated by extensive spectroscopic (IR, MS, 1D and 2D NMR) data analyses. The inhibitory activity of some compounds against a panel of human disease related fungi and bacteria are evaluated. Bioassay for the regulation of tyrosine kinases revealed compounds 1 and 4 possessing selective inhibition against the kinase LCK. Primary structure–activity relationship is discussed.     

Culturable heterotrophic bacteria from the marine sponge <Emphasis Type="Italic">Dendrilla</Emphasis> <Emphasis Type="Italic">nigra</Emphasis>: isolation and phylogenetic diversity of actinobacteria

Culturable heterotrophic bacterial composition of marine sponge Dendrilla nigra was analysed using different enrichments. Five media compositions including without enrichment (control), enriched with sponge extract, with growth regulator (antibiotics), with autoinducers, and complete enrichment containing sponge extract, antibiotics, and autoinducers were developed. DNA hybridization assay was performed to explore host specific bacteria and ecotypes of culturable sponge-associated bacteria. Enrichment with selective inducers (AHLs and sponge extract) and regulators (antibiotics) considerably enhanced the cultivation potential of sponge-associated bacteria. It was found that Marinobacter (MSI032), Micromonospora (MSI033), Streptomyces (MSI051), and Pseudomonas (MSI057) were sponge-associated obligate symbionts. The present findings envisaged that “Micromonospora–Saccharomonospora–Streptomyces” group was the major culturable actinobacteria in the marine sponge D. nigra. The DNA hybridization assay was a reliable method for the analysis of culturable bacterial community in marine sponges. Based on the culturable community structure, the sponge-associated bacteria can be grouped (ecotypes) as general symbionts, specific symbionts, habitat flora, and antagonists.     

Purification and molecular docking study of angiotensin I-converting enzyme (ACE) inhibitory peptides from hydrolysates of marine sponge Stylotella aurantium

Angioteinsin I-converting enzyme (ACE) inhibitory peptide was isolated from marine sponge (Stylotella aurantium) hydrolysate prepared by various hydrolysis enzymes. The peptic hydrolysate exhibited highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight. The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. The amino acid sequences of the purified peptides were identified to be Tyr-Arg (337.2 Da), and Ile-Arg (287.2 Da). The purified peptides from marine sponge had an IC₅₀ value of 237.2 μM and 306.4 μM, respectively. The molecular docking study revealed that ACE inhibitory activity of the purified peptides was mainly attributed to the hydrogen bond interactions and Pi interaction between the dipeptides and ACE. The results suggest that marine sponge, S. aurantium would be an attractive raw material for the manufacture of anti-hypertensive nutraceutical ingredients.     

Phosphocalyculin C as a pyrophosphate protoxin of calyculin C in the marine sponge Discodermia calyx

Calyculin C, a minor derivative of the calyculins, has an additional methyl group on C32 of calyculin A. A recent biosynthetic study of calyculins revealed that an end product of calyculin biosynthesis is the pyrophosphate form, phosphocalyculin A. However, the pyrophosphate counterpart derived from calyculin C had not been reported. We isolated phosphocalyculin C as a minor pyrophosphate derivative, by a detailed investigation of an extract from the sponge Discodermia calyx. The treatment of phosphocalyculin C with the D. calyx cell-free extract significantly enhanced its cytotoxicity, providing molecular evidence for its role as the protoxin of calyculin C.     

Inhibition of Quorum Sensing Mediated Virulence Factors Production in Urinary Pathogen Serratia marcescens PS1 by Marine Sponges

The focal intent of this study was to find out an alternative strategy for the antibiotic usage against bacterial infections. The quorum sensing inhibitory (QSI) activity of marine sponges collected from Palk Bay, India was evaluated against acyl homoserine lactone (AHL) mediated violacein production in Chromobacterium violaceum (ATCC 12472), CV026 and virulence gene expressions in clinical isolate Serratia marcescens PS1. Out of 29 marine sponges tested, the methanol extracts of Aphrocallistes bocagei (TS 8), Haliclona (Gellius) megastoma (TS 25) and Clathria atrasanguinea (TS 27) inhibited the AHL mediated violacein production in C. violaceum (ATCC 12472) and CV026. Further, these sponge extracts inhibited the AHL dependent prodigiosin pigment, virulence enzymes such as protease, hemolysin production and biofilm formation in S. marcescens PS1. However, these sponge extracts were not inhibitory to bacterial growth, which reveals the fact that the QSI activity of these extracts was not related to static or killing effects on bacteria. Based on the obtained results, it is envisaged that the marine sponges could pave the way to prevent quorum sensing (QS) mediated bacterial infections.     

Purification and Characterization of a Collagenolytic Enzyme from a Pathogen of the Great Barrier Reef Sponge,Rhopaloeides odorabile

Background

In recent years there has been a global increase in reports of disease affecting marine sponges. While disease outbreaks have the potential to seriously impact on the survival of sponge populations, the ecology of the marine environment and the health of associated invertebrates, our understanding of sponge disease is extremely limited.

Methodology/Principal Findings

A collagenolytic enzyme suspected to enhance pathogenicity of bacterial strain NW4327 against the sponge Rhopaloeides odorabile was purified using combinations of size exclusion and anion exchange chromatography. After achieving a 77-fold increase in specific activity, continued purification decreased the yield to 21-fold with 7.2% recovery (specific activity 2575 collagen degrading units mg⁻¹protein) possibly due to removal of co-factors. SDS-PAGE of the partially pure enzyme showed two proteins weighing approximately 116 and 45 kDa with the heavier band being similar to reported molecular weights of collagenases from Clostridium and marine Vibrios. The enzyme degraded tissue fibres of several sponge genera suggesting that NW4327 could be deleterious to other sponge species. Activity towards casein and bird feather keratin indicates that the partially purified collagenase is either a non-selective protease able to digest collagen or is contaminated with non-specific proteases. Enzyme activity was highest at pH 5 (the internal pH of R. odorabile) and 30°C (the average ambient seawater temperature). Activity under partially anaerobic conditions also supports the role of this enzyme in the degradation of the spongin tissue. Cultivation of NW4327 in the presence of collagen increased production of collagenase by 30%. Enhanced enzyme activity when NW4327 was cultivated in media formulated in sterile natural seawater indicates the presence of other factors that influence enzyme synthesis.

Conclusions/Significance

Several aspects of the sponge disease etiology were revealed, particularly the strong correlation with the internal tissue chemistry and environmental temperature. This research provides a platform for further investigations into the virulence mechanisms of sponge pathogens.     

N-Formyllapatin A,a new N-formylspiroquinazoline derivative from the marine-derived fungus Penicillium adametzioides AS-53

N-Formyllapatin A (1), a new spiroquinazoline derivative, and four known quinazoline metabolites, lapatins A (2) and B (3), prelapatin B (4), and glyantrypine (5), along with two known indolediketopiperazine derivatives, fumitremorgin B (6) and verruculogen (7), were characterized from Penicillium adametzioides AS-53, a fungus obtained from the fresh tissue of an unidentified marine sponge. The structure of compound 1 was established by detailed interpretation of NMR and MS data, and its absolute configuration was established by a single-crystal X-ray diffraction analysis. N-Formyllapatin A (1) represents the first N-formylspiroquinazoline secondary metabolite. Compounds 3 and 5–7 showed moderate inhibitory activity against aqua-pathogenic bacterial Vibrio harveyi.     

Metagenomic Analysis Reveals Diverse Polyketide Synthase Gene Clusters in Microorganisms Associated with the Marine Sponge Discodermia dissoluta

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up <1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.