Localization and Characterization of Ferritin in Demospongiae: A Possible Role on Spiculogenesis

Iron, as inorganic ion or as oxide, is widely used by biological systems in a myriad of biological functions (e.g., enzymatic, gene activation and/or regulation). In particular, marine organisms containing silica structures—diatoms and sponges—grow preferentially in the presence of iron. Using primary sponge cell culture from S. domuncula–primmorphs—as an in vitro model to study the Demospongiae spiculogenesis, we found the presence of agglomerates 50 nm in diameter exclusively inside sponge specialized cells called sclerocytes. A clear phase/material separation is observed between the agglomerates and the initial stages of intracellular spicule formation. STEM-HRTEM-EDX analysis of the agglomerates (30–100 nm) showed that they are composed of pseudohexagonal nanoparticles between 5 and 15 nm in size, displaying lattice parameters corresponding to hematite (Fe₂O₃) and mixed iron oxide phases typically attributed to ferritin. Further analysis, using western blotting, inductively coupled plasma mass spectrometry (ICP-MS), sequence alignment analysis, immunostaining and magnetic resonance imaging (MRI), of mature spicule filaments confirm the presence of ferritin within these organic structures. We suggest that S. domuncula can be classified as a dual biomineralizating organism, i.e., within the same cellular structure two distinct biomineralizing processes can occur as a result of the same cellular/metabolic function, spiculogenesis.     

Structural Characterization of New Peptide Variants Produced by Cyanobacteria from the Brazilian Atlantic Coastal Forest Using Liquid Chromatography Coupled to Quadrupole Time-of-Flight Tandem Mass Spectrometry

Cyanobacteria from underexplored and extreme habitats are attracting increasing attention in the search for new bioactive substances. However, cyanobacterial communities from tropical and subtropical regions are still largely unknown, especially with respect to metabolite production. Among the structurally diverse secondary metabolites produced by these organisms, peptides are by far the most frequently described structures. In this work, liquid chromatography/electrospray ionization coupled to high resolution quadrupole time-of-flight tandem mass spectrometry with positive ion detection was applied to study the peptide profile of a group of cyanobacteria isolated from the Southeastern Brazilian coastal forest. A total of 38 peptides belonging to three different families (anabaenopeptins, aeruginosins, and cyanopeptolins) were detected in the extracts. Of the 38 peptides, 37 were detected here for the first time. New structural features were proposed based on mass accuracy data and isotopic patterns derived from full scan and MS/MS spectra. Interestingly, of the 40 surveyed strains only nine were confirmed to be peptide producers; all of these strains belonged to the order Nostocales (three Nostoc sp., two Desmonostoc sp. and four Brasilonema sp.).     

Antioxidant and Antimicrobial Potential of the Bifurcaria bifurcata Epiphytic Bacteria

Surface-associated marine bacteria are an interesting source of new secondary metabolites. The aim of this study was the isolation and identification of epiphytic bacteria from the marine brown alga, Bifurcaria bifurcata, and the evaluation of the antioxidant and antimicrobial activity of bacteria extracts. The identification of epiphytic bacteria was determined by 16S rRNA gene sequencing. Bacteria extracts were obtained with methanol and dichloromethane (1:1) extraction. The antioxidant activity of extracts was performed by quantification of total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and oxygen radical absorbance capacity (ORAC). Antimicrobial activities were evaluated against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella enteritidis, Staphylococcus aureus, Saccharomyces cerevisiae and Candida albicans. A total of 39 Bifurcaria bifurcata-associated bacteria were isolated and 33 were identified as Vibrio sp. (48.72%), Alteromonas sp. (12.82%), Shewanella sp. (12.26%), Serratia sp. (2.56%), Citricoccus sp. (2.56%), Cellulophaga sp. (2.56%), Ruegeria sp. (2.56%) and Staphylococcus sp. (2.56%). Six (15.38%) of the 39 bacteria Bifurcaria bifurcata-associated bacteria presented less than a 90% Basic Local Alignment Search Tool (BLAST) match, and some of those could be new. The highest antioxidant activity and antimicrobial activity (against B. subtilis) was exhibited by strain 16 (Shewanella sp.). Several strains also presented high antimicrobial activity against S. aureus, mainly belonging to Alteromonas sp. and Vibrio sp. There were no positive results against fungi and Gram-negative bacteria. Bifurcaria bifurcata epiphytic bacteria were revealed to be excellent sources of natural antioxidant and antimicrobial compounds.     

Briarenolides K and L,New Anti-Inflammatory Briarane Diterpenoids from an Octocoral Briareum sp. (Briareidae)

Two new briarane-type diterpenoids, briarenolides K (1) and L (2), were isolated from an octocoral identified as Briareum sp. The structures of new briaranes 1 and 2 were elucidated by spectroscopic methods. In the in vitro anti-inflammatory effects test, briaranes 1 and 2 were found to significantly inhibit the accumulation of the pro-inflammatory iNOS protein of the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells.     

Molecular and Chemical Analysis of the Lipopolysaccharide from Aeromonas hydrophila Strain AH-1 (Serotype O11)

A group of virulent Aeromonas hydrophila, A. sobria, and A. veronii biovar sobria strains isolated from humans and fish have been described; these strains classified to serotype O11 are serologically related by their lipopolysaccharide (LPS) O-antigen (O-polysaccharide), and the presence of an S-layer consisting of multiple copies of a crystalline surface array protein with a molecular weight of 52 kDa in the form of a crystalline surface array which lies peripheral to the cell wall. A. hydrophila strain AH-1 is one of them. We isolated the LPS from this strain and determined the structure of the O-polysaccharide, which was similar to that previously described for another strain of serotype O11. The genetics of the O11-antigen showed the genes (wb_O11 cluster) in two sections separated by genes involved in biosynthesis and assembly of the S-layer. The O11-antigen LPS is an example of an ABC-2-transporter-dependent pathway for O-antigen heteropolysaccharide (disaccharide) assembly. The genes involved in the biosynthesis of the LPS core (waa_O11 cluster) were also identified in three different chromosome regions being nearly identical to the ones described for A. hydrophila AH-3 (serotype O34). The genetic data and preliminary chemical analysis indicated that the LPS core for strain AH-1 is identical to the one for strain AH-3.     

New and Rare Carotenoids Isolated from Marine Bacteria and Their Antioxidant Activities

Marine bacteria have not been examined as extensively as land bacteria. We screened carotenoids from orange or red pigments-producing marine bacteria belonging to rare or novel species. The new acyclic carotenoids with a C₃₀ aglycone, diapolycopenedioc acid xylosylesters A–C and methyl 5-glucosyl-5,6-dihydro-apo-4,4′-lycopenoate, were isolated from the novel Gram-negative bacterium Rubritalea squalenifaciens, which belongs to phylum Verrucomicrobia, as well as the low-GC Gram-positive bacterium Planococcus maritimus strain iso-3 belonging to the class Bacilli, phylum Firmicutes, respectively. The rare monocyclic C₄₀ carotenoids, (3R)-saproxanthin and (3R,2′S)-myxol, were isolated from novel species of Gram-negative bacteria belonging to the family Flavobacteriaceae, phylum Bacteroidetes. In this review, we report the structures and antioxidant activities of these carotenoids, and consider relationships between bacterial phyla and carotenoid structures.     

Surface Glucan Structures in Aeromonas spp.

Aeromonas spp. are generally found in aquatic environments, although they have also been isolated from both fresh and processed food. These Gram-negative, rod-shaped bacteria are mostly infective to poikilothermic animals, although they are also considered opportunistic pathogens of both aquatic and terrestrial homeotherms, and some species have been associated with gastrointestinal and extraintestinal septicemic infections in humans. Among the different pathogenic factors associated with virulence, several cell-surface glucans have been shown to contribute to colonization and survival of Aeromonas pathogenic strains, in different hosts. Lipopolysaccharide (LPS), capsule and α-glucan structures, for instance, have been shown to play important roles in bacterial–host interactions related to pathogenesis, such as adherence, biofilm formation, or immune evasion. In addition, glycosylation of both polar and lateral flagella has been shown to be mandatory for flagella production and motility in different Aeromonas strains, and has also been associated with increased bacterial adhesion, biofilm formation, and induction of the host proinflammatory response. The main aspects of these structures are covered in this review.     

Intracellular Immunohistochemical Detection of Tetrodotoxin in Pleurobranchaea maculata (Gastropoda) and Stylochoplana sp. (Turbellaria)

Tetrodotoxin (TTX), is a potent neurotoxin targeting sodium channels that has been identified in multiple marine and terrestrial organisms. It was recently detected in the Opisthobranch Pleurobranchaea maculata and a Platyhelminthes Stylochoplana sp. from New Zealand. Knowledge on the distribution of TTX within these organisms is important to assist in elucidating the origin and ecological role of this toxin. Intracellular micro-distribution of TTX was investigated using a monoclonal antibody-based immunoenzymatic technique. Tetrodotoxin was strongly localized in neutral mucin cells and the basement membrane of the mantle, the oocytes and follicles of the gonad tissue, and in the digestive tissue of P. maculata. The ova and pharynx were the only two structures to contain TTX in Stylochoplana sp. Using liquid chromatography-mass spectrometry, TTX was identified in the larvae and eggs, but not the gelatinous egg cases of P. maculata. Tetrodotoxin was present in egg masses of Stylochoplana sp. These data suggest that TTX has a defensive function in adult P. maculata, who then invest this in their progeny for protection. Localization in the digestive tissue of P. maculata potentially indicates a dietary source of TTX. Stylochoplana sp. may use TTX in prey capture and for the protection of offspring.     

Bioactive Phenylalanine Derivatives and Cytochalasins from the Soft Coral-Derived Fungus,Aspergillus elegans

One new phenylalanine derivative 4′-OMe-asperphenamate (1), along with one known phenylalanine derivative (2) and two new cytochalasins, aspochalasin A1 (3) and cytochalasin Z24 (4), as well as eight known cytochalasin analogues (5–12) were isolated from the fermentation broth of Aspergillus elegans ZJ-2008010, a fungus obtained from a soft coral Sarcophyton sp. collected from the South China Sea. Their structures and the relative configurations were elucidated using comprehensive spectroscopic methods. The absolute configuration of 1 was determined by chemical synthesis and Marfey’s method. All isolated metabolites (1–12) were evaluated for their antifouling and antibacterial activities. Cytochalasins 5, 6, 8 and 9 showed strong antifouling activity against the larval settlement of the barnacle Balanus amphitrite, with the EC₅₀ values ranging from 6.2 to 37 μM. This is the first report of antifouling activity for this class of metabolites. Additionally, 8 exhibited a broad spectrum of antibacterial activity, especially against four pathogenic bacteria Staphylococcus albus, S. aureus, Escherichia coli and Bacillus cereus.     

Functional Genomics of the Aeromonas salmonicida Lipopolysaccharide O-Antigen and A-Layer from Typical and Atypical Strains

The A. salmonicida A450 LPS O-antigen, encoded by the wb_salmo gene cluster, is exported through an ABC-2 transporter-dependent pathway. It represents the first example of an O-antigen LPS polysaccharide with three different monosaccharides in their repeating unit assembled by this pathway. Until now, only repeating units with one or two different monosaccharides have been described. Functional genomic analysis of this wb_salmo region is mostly in agreement with the LPS O-antigen structure of acetylated l-rhamnose (Rha), d-glucose (Glc), and 2-amino-2-deoxy-d-mannose (ManN). Between genes of the wb_salmo we found the genes responsible for the biosynthesis and assembly of the S-layer (named A-layer in these strains). Through comparative genomic analysis and in-frame deletions of some of the genes, we concluded that all the A. salmonicida typical and atypical strains, other than A. salmonicida subsp. pectinolytica strains, shared the same wb_salmo and presence of A-layer. A. salmonicida subsp. pectinolytica strains lack wb_salmo and A-layer, two major virulence factors, and this could be the reason they are the only ones not found as fish pathogens.     

Monacyclinones,New Angucyclinone Metabolites Isolated from Streptomyces sp. M7_15 Associated with the Puerto Rican Sponge Scopalina ruetzleri

During an investigation of new actinomycete species from Caribbean sponges for novel bioactive natural products, frigocyclinone (1), dimethyldehydrorabelomycin (3) and six new angucyclinone derivatives were isolated from Streptomyces sp. strain M7_15 associated with the sponge Scopalina ruetzleri. Of these, monacyclinones A–B (4–5) contain the core ring structure of dehydrorabelomycin (2) with the aminodeoxysugar found in frigocyclinone (1). Monacyclinone C (6) is a hydroxylated variant of frigocyclinone (1) and monacyclinone D (7) is a Baeyer Villiger derivative of (6) which also exists as the open chain hydrolysis product monacyclinone E (8). Monacyclinone F (9) contains two unique epoxide rings attached to the angucyclinone moiety and an additional aminodeoxysugar attached through an angular oxygen bond. All structures were confirmed through spectral analyses. Activity against rhabdomycosarcoma cancer cells (SJCRH30) after 48 h of treatment was observed with frigocyclinone (1; EC₅₀ = 5.2 µM), monacyclinone C (6; 160 µM), monacyclinone E (8; 270 µM), and monacyclinone F (9; 0.73 µM). The strongest bioactivity against rhabdomycosarcoma cancer cells and gram-positive bacteria was exhibited by compound 9, suggesting that the extra aminodeoxysugar subunit is important for biological activity.     

Biological Activities of Ethanolic Extracts from Deep-Sea Antarctic Marine Sponges

We report on the screening of ethanolic extracts from 33 deep-sea Antarctic marine sponges for different biological activities. We monitored hemolysis, inhibition of acetylcholinesterase, cytotoxicity towards normal and transformed cells and growth inhibition of laboratory, commensal and clinically and ecologically relevant bacteria. The most prominent activities were associated with the extracts from sponges belonging to the genus Latrunculia, which show all of these activities. While most of these activities are associated to already known secondary metabolites, the extremely strong acetylcholinesterase inhibitory potential appears to be related to a compound unknown to date. Extracts from Tetilla leptoderma, Bathydorus cf. spinosus, Xestospongia sp., Rossella sp., Rossella cf. racovitzae and Halichondria osculum were hemolytic, with the last two also showing moderate cytotoxic potential. The antibacterial tests showed significantly greater activities of the extracts of these Antarctic sponges towards ecologically relevant bacteria from sea water and from Arctic ice. This indicates their ecological relevance for inhibition of bacterial microfouling.     

Rosmarinic Acid from Eelgrass Shows Nematicidal and Antibacterial Activities against Pine Wood Nematode and Its Carrying Bacteria

Pine wilt disease (PWD), a destructive disease for pine trees, is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus and additional bacteria. In this study, extracts of Zostera marina showed a high nematicidal activity against PWN and some of the bacteria that it carries. Light yellow crystals were obtained from extracts of Z. marina through solvent extraction, followed by chromatography on AB-8 resin and crystallization. The NMR and HPLC analysis showed that the isolated compound was rosmarinic acid (RosA). RosA showed effective nematicidal activity, of which the LC₅₀ (50% lethal concentration) to PWN at 24 h, 48 h and 72 h was 1.18 mg/g, 1.05 mg/g and 0.95 mg/g, respectively. To get a high yield rate of RosA from Z. marina, single factor experiments and an L₉ (3⁴) orthogonal experiment were performed. This extraction process involved 70% ethanol for 3 h at 40 °C. The extraction dosage was 1:50 (w/v). The highest yield of RosA from Zostera was 3.13 mg/g DW (dried weight). The crude extracts of Zostera marina (10 mg/mL) and RosA (1 mg/mL) also showed inhibitory effects to some bacterial strains carried by PWN: Klebsiella sp., Stenotrophomonas maltophilia, Streptomyces sp. and Pantoea agglomerans. The results of these studies provide clues for preparing pesticide to control PWD from Z. marina.     

Briarenolides U–Y,New Anti-Inflammatory Briarane Diterpenoids from an Octocoral Briareum sp. (Briareidae)

Five new 13,14-epoxybriarane diterpenoids, briarenolides U–Y (1–5), were isolated from the octocoral Briareum sp. The structures of briaranes 1–5 were elucidated by spectroscopic methods. Briarenolides U–Y (1–5) were found to significantly inhibit the expression of the pro-inflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein of the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells.     

Dereplication Strategies for Targeted Isolation of New Antitrypanosomal Actinosporins A and B from a Marine Sponge Associated-Actinokineospora sp. EG49

High resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy were employed as complementary metabolomic tools to dereplicate the chemical profile of the new and antitrypanosomally active sponge-associated bacterium Actinokineospora sp. EG49 extract. Principal Component (PCA), hierarchical clustering (HCA), and orthogonal partial least square-discriminant analysis (OPLS-DA) were used to evaluate the HRFTMS and NMR data of crude extracts from four different fermentation approaches. Statistical analysis identified the best culture one-strain-many-compounds (OSMAC) condition and extraction procedure, which was used for the isolation of novel bioactive metabolites. As a result, two new O-glycosylated angucyclines, named actinosporins A (1) and B (2), were isolated from the broth culture of Actinokineospora sp. strain EG49, which was cultivated from the Red Sea sponge Spheciospongia vagabunda. The structures of actinosporins A and B were determined by 1D- and 2D-NMR techniques, as well as high resolution tandem mass spectrometry. Testing for antiparasitic properties showed that actinosporin A exhibited activity against Trypanosoma brucei brucei with an IC₅₀ value of 15 µM; however no activity was detected against Leishmania major and Plasmodium falciparum, therefore suggesting its selectivity against the parasite Trypanosoma brucei brucei; the causative agent of sleeping sickness.     

Capgermacrenes A and B,Bioactive Secondary Metabolites from a Bornean Soft Coral,Capnella sp.

Two new bicyclogermacrenes, capgermacrenes A (1) and B (2), were isolated with two known compounds, palustrol (3) and litseagermacrane (4), from a population of Bornean soft coral Capnella sp. The structures of these metabolites were elucidated based on spectroscopic data. Compound 1 was found to inhibit the accumulation of the LPS-induced pro-inflammatory IL-1β and NO production by down-regulating the expression of iNOS protein in RAW 264.7 macrophages.     

Construction of Escherichia coli Mutant with Decreased Endotoxic Activity by Modifying Lipid A Structure

Escherichia coli BL21 (DE3) and its derivatives are widely used for the production of recombinant proteins, but these purified proteins are always contaminated with lipopolysaccharide (LPS). LPS is recognized by the toll-like receptor 4 and myeloid differentiation factor 2 complex of mammalian immune cells and leads to release of pro-inflammatory cytokines. It is a vital step to remove LPS from the proteins before use for therapeutic purpose. In this study, we constructed BL21 (DE3) ∆msbB28 ∆pagP38 mutant, which produces a penta-acylated LPS with reduced endotoxicity. The plasmids harboring pagL and/or lpxE were then introduced into this mutant to further modify the LPS. The new strain (S004) carrying plasmid pQK004 (pagL and lpxE) produced mono-phosphoryated tetra-acylated lipid A, which induces markedly less production of tumor necrosis factor-α in the RAW264.7 and IL-12 in the THP1, but still retains ability to produce recombinant proteins. This study provides a strategy to decrease endotoxic activity of recombinant proteins purified from E. coli BL21 backgrounds and a feasible approach to modify lipid A structure for alternative purposes such as mono-phosphoryl lipid A (MPL) as vaccine adjuvants.     

Structural and Immunochemical Studies of the Lipopolysaccharide from the Fish Pathogen,Aeromonas bestiarum Strain K296, Serotype O18

Chemical analyses and mass spectrometry were used to study the structure of the lipopolysaccharide (LPS) isolated from Aeromonas bestiarum strain K296, serotype O18. ESI-MS revealed that the most abundant A. bestiarum LPS glycoforms have a hexa-acylated or tetra-acylated lipid A with conserved architecture of the backbone, consisting of a 1,4′-bisphosphorylated β-(1→6)-linked d-GlcN disaccharide with an AraN residue as a non-stoichiometric substituent and a core oligosaccharide composed of Kdo₁Hep₆Hex₁HexN₁P₁. 1D and 2D NMR spectroscopy revealed that the O-specific polysaccharide (OPS) of A. bestiarum K296 consists of a branched tetrasaccharide repeating unit containing two 6-deoxy-l-talose (6dTalp), one Manp and one GalpNAc residues; thus, it is similar to that of the OPS of A. hydrophila AH-3 (serotype O34) in both the sugar composition and the glycosylation pattern. Moreover, 3-substituted 6dTalp was 2-O-acetylated and additional O-acetyl groups were identified at O-2 and O-4 (or O-3) positions of the terminal 6dTalp. Western blots with polyclonal rabbit sera showed that serotypes O18 and O34 share some epitopes in the LPS. The very weak reaction of the anti-O34 serum with the O-deacylated LPS of A. bestiarum K296 might have been due to the different O-acetylation pattern of the terminal 6dTalp. The latter suggestion was further confirmed by NMR.     

Identification of a 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid Reductase,FlRed, in an Alginolytic Bacterium Flavobacterium sp. Strain UMI-01

In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11). The monosaccharide is non-enzymatically converted to 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH), then reduced to 2-keto-3-deoxy-d-gluconate (KDG) by a specific reductase, and metabolized through the Entner–Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR) superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%–88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed) was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.