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.     

Structural Studies of the Lipopolysaccharide from the Fish Pathogen Aeromonas veronii Strain Bs19, Serotype O16

Chemical analyses, mass spectrometry, and NMR spectroscopy were applied to study the structure of the lipopolysaccharide (LPS) isolated from Aeromonas veronii strain Bs19, serotype O16. ESI-MS revealed that the most abundant LPS glycoforms have tetra-acylated or hexa-acylated lipid A species, consisting of a bisphosphorylated GlcN disaccharide with an AraN residue as a non-stoichiometric substituent, and a core oligosaccharide composed of Hep₅Hex₃HexN₁Kdo₁P₁. Sugar and methylation analysis together with 1D and 2D ¹H and ¹³C NMR spectroscopy were the main methods used, and revealed that the O-specific polysaccharide (OPS) of A. veronii Bs19 was built up of tetrasaccharide repeating units with the structure: →4)-α-d-Quip3NAc-(1→3)-α-l-Rhap-(1→4)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→. This composition was confirmed by mass spectrometry. The charge-deconvoluted ESI FT-ICR MS recorded for the LPS preparations identified mass peaks of SR- and R-form LPS species, that differed by Δm = 698.27 u, a value corresponding to the calculated molecular mass of one OPS repeating unit (6dHexNAc6dHexHexHexNAc-H₂O). Moreover, unspecific fragmentation spectra confirmed the sequence of the sugar residues in the OPS and allowed to assume that the elucidated structure also represented the biological repeating unit.     

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.     

The Lipopolysaccharide Export Pathway in Escherichia coli: Structure,Organization and Regulated Assembly of the Lpt Machinery

The bacterial outer membrane (OM) is a peculiar biological structure with a unique composition that contributes significantly to the fitness of Gram-negative bacteria in hostile environments. OM components are all synthesized in the cytosol and must, then, be transported efficiently across three compartments to the cell surface. Lipopolysaccharide (LPS) is a unique glycolipid that paves the outer leaflet of the OM. Transport of this complex molecule poses several problems to the cells due to its amphipatic nature. In this review, the multiprotein machinery devoted to LPS transport to the OM is discussed together with the challenges associated with this process and the solutions that cells have evolved to address the problem of LPS biogenesis.     

Influence of Core Oligosaccharide of Lipopolysaccharide to Outer Membrane Behavior of Escherichia coli

Lipopolysaccharides, major molecules in the outer membrane of Gram-negative bacteria, play important roles on membrane integrity of the cell. However, how the core oligosaccharide of lipopolysaccharide affect the membrane behavior is not well understood. In this study, the relationship between the core oligosaccharide of lipopolysaccharide and the membrane behavior was investigated using a series of Escherichia coli mutants defective in genes to affect the biosynthesis of core oligosaccharide of lipopolysaccharide. Cell surface hydrophobicity, outer membrane permeability, biofilm formation and auto-aggregation of these mutant cells were compared. Compared to the wild type W3110, cell surface hydrophobicities of mutant ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaP, ΔwaaY and ΔwaaB were enhanced, outer membrane permeabilities of ΔwaaC, ΔwaaF, ΔwaaG and ΔwaaP were significantly increased, abilities of biofilm formation by ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaR, ΔwaaP, ΔwaaQ and ΔwaaY decreased, and auto-aggregation abilities of ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaR, ΔwaaU, ΔwaaP and ΔwaaY were strongly enhanced. These results give new insight into the influence of core oligosaccharide of lipopolysaccharide on bacterial cell membrane behavior.     

Lipopolysaccharides from Commensal and Opportunistic Bacteria: Characterization and Response of the Immune System of the Host Sponge Suberites domuncula

Marine sponges harbor a rich bacterioflora with which they maintain close relationships. However, the way these animals make the distinction between bacteria which are consumed to meet their metabolic needs and opportunistic and commensal bacteria which are hosted is not elucidated. Among the elements participating in this discrimination, bacterial cell wall components such as lipopolysaccharides (LPS) could play a role. In the present study, we investigated the LPS chemical structure of two bacteria associated with the sponge Suberites domuncula: a commensal Endozoicomonas sp. and an opportunistic Pseudoalteromonas sp. Electrophoretic patterns indicated different LPS structures for these bacteria. The immunomodulatory lipid A was isolated after mild acetic acid hydrolysis. The electrospray ionization ion-trap mass spectra revealed monophosphorylated molecules corresponding to tetra- and pentaacylated structures with common structural features between the two strains. Despite peculiar structural characteristics, none of these two LPS influenced the expression of the macrophage-expressed gene S. domuncula unlike the Escherichia coli ones. Further research will have to include a larger number of genes to understand how this animal can distinguish between LPS with resembling structures and discriminate between bacteria associated with it.     

Erylusamides: Novel Atypical Glycolipids from Erylus cf. deficiens

Among marine organisms, sponges are the richest sources of pharmacologically-active compounds. Stemming from a previous lead discovery program that gathered a comprehensive library of organic extracts of marine sponges from the off-shore region of Portugal, crude extracts of Erylus cf. deficiens collected in the Gorringe Bank (Atlantic Ocean) were tested in the innovative high throughput screening (HTS) assay for inhibitors of indoleamine 2,3-dioxygenase (IDO) and showed activity. Bioassay guided fractionation of the dichloromethane extract led to the isolation of four new glycolipids, named erylusamide A–D. The structures of the isolated compounds were established by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and chemical derivatization. The metabolites shared a pentasaccharide moiety constituted by unusual highly acetylated d-glucose moieties as well as d-xylose and d-galactose. The aglycones were unprecedented long chain dihydroxyketo amides. Erylusamides A, B and D differ in the length of the hydrocarbon chain, while erylusamide C is a structural isomer of erylusamide B.     

Structural Investigation of the Oligosaccharide Portion Isolated from the Lipooligosaccharide of the Permafrost Psychrophile Psychrobacter arcticus 273-4

Psychrophilic microorganisms have successfully colonized all permanently cold environments from the deep sea to mountain and polar regions. The ability of an organism to survive and grow in cryoenviroments depends on a number of adaptive strategies aimed at maintaining vital cellular functions at subzero temperatures, which include the structural modifications of the membrane. To understand the role of the membrane in the adaptation, it is necessary to characterize the cell-wall components, such as the lipopolysaccharides, that represent the major constituent of the outer membrane. The aim of this study was to investigate the structure of the carbohydrate backbone of the lipooligosaccharide (LOS) isolated from the cold-adapted Psychrobacter arcticus 273-4. The strain, isolated from a 20,000-to-30,000-year-old continuously frozen permafrost in Siberia, was cultivated at 4 °C. The LOS was isolated from dry cells and analyzed by means of chemical methods. In particular, it was degraded either by mild acid hydrolysis or by hydrazinolysis and investigated in detail by ¹H and ¹³C NMR spectroscopy and by ESI FT-ICR mass spectrometry. The oligosaccharide was characterized by the substitution of the heptose residue, usually linked to Kdo in the inner core, with a glucose, and for the unusual presence of N-acetylmuramic acid.     

Identification of Two Novel Anti-Fibrotic Benzopyran Compounds Produced by Engineered Strains Derived from Streptomyces xiamenensis M1-94P that Originated from Deep-Sea Sediments

The benzopyran compound obtained by cultivating a mangrove-derived strain, Streptomyces xiamenensis strain 318, shows multiple biological effects, including anti-fibrotic and anti-hypertrophic scar properties. To increase the diversity in the structures of the available benzopyrans, by means of biosynthesis, the strain was screened for spontaneous rifampicin resistance (Rif), and a mutated rpsL gene to confer streptomycin resistance (Str), was introduced into the S. xiamenensis strain M1-94P that originated from deep-sea sediments. Two new benzopyran derivatives, named xiamenmycin C (1) and D (2), were isolated from the crude extracts of a selected Str-Rif double mutant (M6) of M1-94P. The structures of 1 and 2 were identified by analyzing extensive spectroscopic data. Compounds 1 and 2 both inhibit the proliferation of human lung fibroblasts (WI26), and 1 exhibits better anti-fibrotic activity than xiamenmycin. Our study presents the novel bioactive compounds isolated from S. xiamenensis mutant strain M6 constructed by ribosome engineering, which could be a useful approach in the discovery of new anti-fibrotic compounds.     

Oxygenated Eremophilane- and Neolemnane-Derived Sesquiterpenoids from the Soft Coral Lemnalia philippinensis

Five sesquiterpene-related metabolites (1–5), including two new eremophilane-type compounds, philippinlins C and D (1 and 2) and a 4,5-seconeolemnane philippinlin E (3), were isolated from the organic extract of a Taiwanese soft coral Lemnalia philippinensis. The structures of the new metabolites were determined on the basis of extensive spectroscopic analysis and by comparison of NMR data with those of related metabolites. Compound 3 was suggested to be derived from the neolemnane skeleton.     

Gene Sequence Based Clustering Assists in Dereplication of Pseudoalteromonas luteoviolacea Strains with Identical Inhibitory Activity and Antibiotic Production

Some microbial species are chemically homogenous, and the same secondary metabolites are found in all strains. In contrast, we previously found that five strains of P. luteoviolacea were closely related by 16S rRNA gene sequence but produced two different antibiotic profiles. The purpose of the present study was to determine whether such bioactivity differences could be linked to genotypes allowing methods from phylogenetic analysis to aid in selection of strains for biodiscovery. Thirteen P. luteoviolacea strains divided into three chemotypes based on production of known antibiotics and four antibacterial profiles based on inhibition assays against Vibrio anguillarum and Staphylococcus aureus. To determine whether chemotype and inhibition profile are reflected by phylogenetic clustering we sequenced 16S rRNA, gyrB and recA genes. Clustering based on 16S rRNA gene sequences alone showed little correlation to chemotypes and inhibition profiles, while clustering based on concatenated 16S rRNA, gyrB, and recA gene sequences resulted in three clusters, two of which uniformly consisted of strains of identical chemotype and inhibition profile. A major time sink in natural products discovery is the effort spent rediscovering known compounds, and this study indicates that phylogeny clustering of bioactive species has the potential to be a useful dereplication tool in biodiscovery efforts.     

Antinociceptive and anti-inflammatory activity from algae of the genus Caulerpa

Marine natural products have been the focus of discovery for new products of chemical and pharmacological interest. The aim of this study was to evaluate the antinociceptive activity of the methanolic (ME), acetate (AE), hexanic (HE) and chloroform (CE) extracts obtained from Caulerpa mexicana, and ME, CE and HE obtained from Caulerpa sertularioides. These marine algae are found all over the world, mainly in tropical regions. Models such as the writhing test, the hot plate test and formalin-induced nociception test were used to evaluate antinociceptive activity in laboratory mice. In the writhing test, all the extracts were administered orally at a concentration of 100 mg/kg, and induced high peripheral antinociceptive activity, with a reduction in the nociception induced by acetic acid above 65%. In the hot plate test, treatment with extracts from C. sertularioides (100 mg/kg, p.o.) did not significantly increase the latency of response, although the ME, AE and HE from C. mexicana showed activity in this model. This result suggests that these extracts exhibit antinociceptive activity. In the formalin test, it was observed that ME, AE and HE obtained from C. mexicana reduced the effects of formalin in both phases. On the other hand only CE from C. sertularioides induced significant inhibition of the nociceptive response in the first phase. To better assess the potential anti-inflammatory activity of the extracts, the carrageenan-induced peritonitis test was used to test Caulerpa spp. extracts on cell migration into the peritoneal cavity. In this assay, all extracts evaluated were able to significantly inhibit leukocyte migration into the peritoneal cavity in comparison with carrageenan. These data demonstrate that extracts from Caulerpa species elicit pronounced antinociceptive and anti-inflamatory activity against several nociception models. However, pharmacological and chemical studies are continuing in order to characterize the mechanism(s) responsible for the antinociceptive action and also to identify the active principles present in the Caulerpa species.     

Functional Recombinants Designed from a Fetuin/Asialofetuin-Specific Marine Algal Lectin,Rhodobindin

Plant lectins have attracted much attention for biomedical applications including targeted drug delivery system and therapy against tumors and microbial infections. The main problem of using lectins as a biomedical tool is a batch-to-batch variation in isoforms content. The production of lectins using recombination tools has the advantage of obtaining high amounts of proteins with more precise properties, but there are only a handful of functional recombinant lectins presently available. A fetuin/asialo-fetuin specific lectin, Rhodobindin, has unique tandem repeats structure which makes it useful in exploiting for recombinant lectin. We developed three functional recombinant lectins using E. coli expression system: one from full cDNA sequence and two from fragmentary sequences of Rhodobindin. Hemagglutinating activity and solubility of the recombinant lectins were highest at OD 0.7 cell concentration at 20 °C. The optimized process developed in this study was suitable for the quality-controlled production of high amounts of soluble recombinant lectins.     

Isolation and Analysis of the Cppsy Gene and Promoter from Chlorella protothecoides CS-41

Phytoene synthase (PSY) catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, the first colorless carotene in the carotenoid biosynthesis pathway. So it is regarded as the crucial enzyme for carotenoid production, and has unsurprisingly been involved in genetic engineering studies of carotenoid production. In this study, the psy gene from Chlorella protothecoides CS-41, designated Cppsy, was cloned using rapid amplification of cDNA ends. The full-length DNA was 2488 bp, and the corresponding cDNA was 1143 bp, which encoded 380 amino acids. Computational analysis suggested that this protein belongs to the Isoprenoid_Biosyn_C1 superfamily. It contained the consensus sequence, including three predicted substrate-Mg²⁺ binding sites. The Cppsy gene promoter was also cloned and characterized. Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA)-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box. Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA. These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.     

Pigmentation and spectral absorbance signatures in deep-water corals from the Trondheimsfjord, Norway

The pigmentation and corresponding in vivo and in vitro absorption characteristics in three different deep-water coral species: white and orange Lophelia pertusa, Paragorgia arborea and Primnoa resedaeformis, collected from the Trondheimsfjord are described. Pigments were isolated and characterized by High-Performance Liquid Chromatography (HPLC) analysis and High-Performance Liquid Chromatography Time-Of-Flight Mass Spectrometer (LC-TOF MS). The main carotenoids identified for all three coral species were astaxanthin and a canthaxanthin-like carotenoid. Soft tissue and skeleton of orange L. pertusa contained 2 times more astaxanthin g(-1) wet weight compared to white L. pertusa. White and orange L. pertusa were characterized with in vivo absorbance peaks at 409 and 473 nm, respectively. In vivo absorbance maxima for P. arborea and P. resedaeformis was typically at 475 nm. The shapes of the absorbance spectra (400-700 nm) were species-specific, indicated by in vivo, in vitro and the corresponding difference spectra. The results may provide important chemotaxonomic information for pigment when bonded to their proteins in vivo, bio-prospecting, and for in situ identification, mapping and monitoring of corals.     

Indole Diterpenoids and Isocoumarin from the Fungus,Aspergillus flavus,Isolated from the Prawn,Penaeus vannamei

Two new indole-diterpenoids (1 and 2) and a new isocoumarin (3), along with the known β-aflatrem (4), paspalinine (5), leporin B (6), α-cyclopiazonic acid (7), iso-α-cyclopiazonic acid (8), ditryptophenaline (9), aflatoxin B₁ (10), 7-O-acetylkojic acid (11) and kojic acid (12), were isolated from the fermentation broth of the marine-derived fungus, Aspergillus flavus OUCMDZ-2205. The structures of Compounds 1–12 were elucidated by spectroscopic analyses, quantum ECD calculations and the chemical method. New Compound 1 exhibited antibacterial activity against Staphylococcus aureus with a MIC value of 20.5 μM. Both new Compounds 1 and 2 could arrest the A549 cell cycle in the S phase at a concentration of 10 μM. Compound 1 showed PKC-beta inhibition with an IC₅₀ value of 15.6 μM. In addition, the absolute configurations of the known compounds, 4–6 and leporin A (6a), were also determined for the first time.     

Complex Toxin Profile of French Mediterranean Ostreopsis cf. ovata Strains,Seafood Accumulation and Ovatoxins Prepurification

Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative palytoxin (p-PLTX), which can accumulate in marine organisms and may possibly lead to food intoxication. However, purified ovatoxins are not widely available and their toxicities are still unknown. The aim of this study was to improve understanding of the ecophysiology of Ostreopsis cf. ovata and its toxin production as well as to optimize the purification process for ovatoxin. During Ostreopsis blooms in 2011 and 2012 in Villefranche-sur-Mer (France, NW Mediterranean Sea), microalgae epiphytic cells and marine organisms were collected and analyzed both by LC-MS/MS and hemolysis assay. Results obtained with these two methods were comparable, suggesting ovatoxins have hemolytic properties. An average of 223 μg·kg⁻¹ of palytoxin equivalent of whole flesh was found, thus exceeding the threshold of 30 μg·kg⁻¹ in shellfish recommended by the European Food Safety Authority (EFSA). Ostreopsis cells showed the same toxin profile both in situ and in laboratory culture, with ovatoxin-a (OVTX-a) being the most abundant analogue (~50%), followed by OVTX-b (~15%), p-PLTX (12%), OVTX-d (8%), OVTX-c (5%) and OVTX-e (4%). Ostreopsis cf. ovata produced up to 2 g of biomass per L of culture, with a maximum concentration of 300 pg PLTX equivalent cell⁻¹. Thus, an approximate amount of 10 mg of PLTX-group toxins may be produced with 10 L of this strain. Toxin extracts obtained from collected biomass were purified using different techniques such as liquid-liquid partition or size exclusion. Among these methods, open-column chromatography with Sephadex LH20 phase yielded the best results with a cleanup efficiency of 93% and recovery of about 85%, representing an increase of toxin percentage by 13 fold. Hence, this purification step should be incorporated into future isolation exercises.     

Antifungal activity evaluation of the constituents of Haliclona baeri and Haliclona cymaeformis, collected from the Gulf of Thailand

A new compound maleimide-5-oxime was isolated, together with 3,4-dihydroxybenzoic acid, tetillapyrone, from the ethyl acetate extract of the marine sponge Haliclona baeri while tetillapyrone, nortetillapyrone, p-hydroxybenzaldehyde and phenylacetic acid were isolated from the ethyl acetate extract of Haliclona cymaeformis, collected from the Gulf of Thailand. The structures of tetillapyrone and nortetillapyrone were re-examined using HMBC correlations. Maleimide-5-oxime, tetillapyrone and nortetillapyrone were found to be inactive against three human tumor cell lines (the estrogen-dependent ER(+) MCF-7, the estrogen-independent ER(−) MDA-MB-231 and NCI-H460. Maleimide-5-oxime, p-hydroxybenzaldehyde, phenylacetic acid, tetillapyrone and nortetillapyrone were evaluated for their growth inhibitory effect against seven yeasts and eight filamentous fungi. Only nortetillapyrone showed antifungal activity, with a preponderance on the dermatophytic filamentous fungi.     

Terpenoids from the octocorals Menella sp. (Plexauridae) and Lobophytum crassum (Alcyonacea)

A new germacrane-type sesquiterpenoid, menelloide E (1), and a new cembrane-type diterpenoid, lobocrassin F (2), were isolated from the octocorals Menella sp. and Lobophytum crassum, respectively. The structures of terpenoids 1 and 2 were determined by spectroscopic and chemical methods and compound 2 was found to display a significant inhibitory effect on the release of elastase by human neutrophils.