Increasing outer membrane complexity: the case of the lipopolysaccharide lipid A from marine Cellulophaga pacifica.

Gram-negative bacteria living in marine waters have evolved peculiar adaptation strategies to deal with the numerous stress conditions that characterize aquatic environments. Among the multiple mechanisms for efficient adaptation, these bacteria typically exhibit chemical modifications in the structure of the lipopolysaccharide (LPS), which is a fundamental component of their outer membrane. In particular, the glycolipid anchor to the membrane of marine bacteria LPSs, i.e. the lipid A, frequently shows unusual chemical structures, which are reflected in equally singular immunological properties with potential applications as immune adjuvants or anti-sepsis drugs. In this work, we determined the chemical structure of the lipid A from Cellulophaga pacifica KMM 3664T isolated from the Sea of Japan. This bacterium showed to produce a heterogeneous mixture of lipid A molecules that mainly display five acyl chains and carry a single phosphate and a D-mannose disaccharide on the glucosamine backbone. Furthermore, we proved that C. pacifica KMM 3664T LPS acts as a weaker activator of Toll-like receptor 4 (TLR4) compared to the prototypical enterobacterial Salmonella typhimurium LPS. Our results are relevant to the future development of novel vaccine adjuvants and immunomodulators inspired by marine LPS chemistry.

Stonikacidin A, an Antimicrobial 4-Bromopyrrole Alkaloid Containing L-Idonic Acid Core from the Northwestern Pacific Marine Sponge Lissodendoryx papillosa.

Stonikacidin A (1), the first representative of a new class of 4-bromopyrrole alkaloids containing an aldonic acid core, was isolated from the marine sponge Lissodendoryx papillosa. The compound is named in honor of Prof. Valentin A. Stonik, who is one of the outstanding investigators in the field of marine natural chemistry. The structure of 1 was determined using NMR, MS analysis, and chemical correlations. The L-idonic acid core was established by the comparison of GC, NMR, MS, and optical rotation data of methyl-pentaacetyl-aldonates obtained from the hydrolysis products of 1 and standard hexoses. The L-form of the idonic acid residue in 1 was confirmed by GC analysis of pentaacetate of (S)-2-butyl ester of the hydrolysis product from 1 and compared with corresponding derivatives of L- and D-idonic acids. The biosynthetic pathway for stonikacidin A (1) was proposed. The alkaloid 1 inhibited the growth of Staphylococcus aureus and Escherichia coli test strains, as well as affected the formation of S. aureus and E. coli biofilms. Compound 1 inhibited the activity of sortase A. Molecular docking data showed that stonikacidin A (1) can bind with sortase A due to the interactions between its bromine atoms and some amino acid residues of the enzyme.

Pushing the Boundaries of Structural Heterogeneity with the Lipid A of Marine Bacteria Cellulophaga.

Marine bacteria, which are often described as chemical gold, are considered an exceptional source of new therapeutics. Considerable research interest has been given to lipopolysaccharides (LPSs), the main components of the Gram-negative outer membrane. LPS and its lipid A portion from marine bacteria are known to exhibit a tricky chemistry that has been often associated with intriguing properties such as behaving as immune adjuvants or anti-sepsis molecules. In this scenario, we report the structural determination of the lipid A from three marine bacteria within the Cellulophaga genus, which showed to produce an extremely heterogenous blend of tetra- to hexa-acylated lipid A species, mostly carrying one phosphate and one D-mannose on the glucosamine disaccharide backbone. The ability of the three LPSs in activating TLR4 signaling revealed a weaker immunopotential by C. baltica NNO 15840T and C. tyrosinoxydans EM41T , while C. algicola ACAM 630T behaved as a more potent TLR4 activator.

Structural characterization and physicochemical properties of the exopolysaccharide produced by the moderately halophilic bacterium Chromohalobacter salexigens, strain 3EQS1.

A strain, 3EQS1, was isolated from a salt sample taken from Lake Qarun (Fayoum Province, Egypt). On the basis of physiological, biochemical, and phylogenetic analyses, the strain was classified as Chromohalobacter salexigens. By 72 h of growth at 25 °C, strain 3EQS1 produced large amounts (15.1 g L-1) of exopolysaccharide (EPS) in a liquid mineral medium (initial pH 8.0) containing 10% sucrose and 10% NaCl. The EPS was precipitated from the cell-free culture medium with chilled ethanol and was purified by gel-permeation and anion-exchange chromatography. The molecular mass of the EPS was 0.9 × 106 Da. Chemical analyses, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy showed that the EPS was a linear ß-D-(2 → 6)-linked fructan (levan). In aqueous solution, the EPS tended to form supramolecular aggregates with a critical aggregation concentration of 240 µg mL-1. The EPS had high emulsifying activity (E24, %) against kerosene (31.2 ± 0.4%), sunflower oil (76.9 ± 1.3%), and crude oil (98.9 ± 0.8%), and it also had surfactant properties. A 0.1% (w/v) aqueous EPS solution reduced the surface tension of water by 11.9%. The levan of C. salexigens 3EQS1 may be useful in various biotechnological processes.

Molecular Cloning and Characteristics of a Lectin from the Bivalve Glycymeris yessoensis.

C-type lectins (CTLs) are a family of carbohydrate-binding proteins that mediate multiple biological events, including adhesion between cells, the turnover of serum glycoproteins, and innate immune system reactions to prospective invaders. Here, we describe the cDNA cloning of lectin from the bivalve Glycymeris yessoensis (GYL), which encodes 161 amino acids and the C-type carbohydrate recognition domain (CRD) with EPN and WND motifs. The deduced amino acid sequence showed similarity to other CTLs. GYL is a glycoprotein containing two N-glycosylation sites per subunit. N-glycans are made up of xylose, mannose, D-glucosamine, 3-O-methylated galactose, D-quinovoses, and 3-O-methylated 6-deoxy-D-glucose. The potential CRD tertiary structure of the GYL adopted CTL-typical long-form double-loop structure and included three disulfide bridges at the bases of the loops. Additionally, when confirming the GYL sequence, eight isoforms of this lectin were identified. This fact indicates the presence of a multigene family of GYL-like C-type lectins in the bivalve G. yessoensis. Using the glycan microarray approach, natural carbohydrate ligands were established, and the glycotope for GYL was reconstructed as "Galß1-4GlcNAcß obligatory containing an additional fragment", like a sulfate group or a methyl group of fucose or N-acetylgalactosamine residues.

Structure of the Lipooligosaccharide from the Deep-Sea Marine Bacterium Idiomarina zobellii KMM 231T, Isolated at a Depth of 4000 Meters.

The structural characterization of lipopolysaccharides has critical implications for some biomedical applications, and marine bacteria are an inimitable source of new glyco-structures potentially usable in medicinal chemistry. On the other hand, lipopolysaccharides of marine Gram-negative bacteria present certain structural features that can help the understanding of the adaptation processes. The deep-sea marine Gram-negative bacterium Idiomarina zobellii KMM 231T, isolated from a seawater sample taken at a depth of 4000 m, represents an engaging microorganism to investigate in terms of its cell wall components. Here, we report the structural study of the R-type lipopolysaccharide isolated from I. zobellii KMM 231T that was achieved through a multidisciplinary approach comprising chemical analyses, NMR spectroscopy, and MALDI mass spectrometry. The lipooligosaccharide turned out to be characterized by a novel and unique pentasaccharide skeleton containing a very short mono-phosphorylated core region and comprising terminal neuraminic acid. The lipid A was revealed to be composed of a classical disaccharide backbone decorated by two phosphate groups and acylated by i13:0(3-OH) in amide linkage, i11:0 (3-OH) as primary ester-linked fatty acids, and i11:0 as a secondary acyl chain.

Structure of the Cell-Wall-Associated Polysaccharides from the Deep-Sea Marine Bacterium Devosia submarina KMM 9415T.

Two cell-wall-associated polysaccharides were isolated and purified from the deep-sea marine bacterium Devosia submarina KMM 9415T, purified by ultracentrifugation and enzymatic treatment, separated by chromatographic techniques, and studied by sugar analyses and NMR spectroscopy. The first polysaccharide with a molecular weight of about 20.7 kDa was found to contain d-arabinose, and the following structure of its disaccharide repeating unit was established: →2)-α-d-Araf-(1→5)-α-d-Araf-(1→. The second polysaccharide was shown to consist of d-galactose and a rare component of bacterial glycans-d-xylulose: →3)-α-d-Galp-(1→3)-ß-d-Xluf-(1→.

Structure of the 4-O-[1-Carboxyethyl]-d-Mannose-Containing O-Specific Polysaccharide of a Halophilic Bacterium Salinivibrio sp. EG9S8QL.

The moderately halophilic strain Salinivibrio sp. EG9S8QL was isolated among 11 halophilic strains from saline mud (Emisal Salt Company, Lake Qarun, Fayoum, Egypt). The lipopolysaccharide was extracted from dried cells of Salinivibrio sp. EG9S8QL by the phenol-water procedure. The OPS was obtained by mild acid hydrolysis of the lipopolysaccharide and was studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments. The OPS was found to be composed of linear tetrasaccharide repeating units of the following structure: →2)-ß-Manp4Lac-(1→3)-α-ManpNAc-(1→3)-ß-Rhap-(1→4)-α-GlcpNAc-(1→, where Manp4Lac is 4-O-[1-carboxyethyl]mannose.

Structure and in vitro Bioactivity against Cancer Cells of the Capsular Polysaccharide from the Marine Bacterium Psychrobacter marincola.

Psychrobacter marincola KMM 277T is a psychrophilic Gram-negative bacterium that has been isolated from the internal tissues of an ascidian Polysyncraton sp. Here, we report the structure of the capsular polysaccharide from P. marincola KMM 277T and its effect on the viability and colony formation of human acute promyelocytic leukemia HL-60 cells. The polymer was purified by several separation methods, including ultracentrifugation and chromatographic procedures, and the structure was elucidated by means of chemical analysis, 1-D, and 2-D NMR spectroscopy techniques. It was found that the polysaccharide consists of branched hexasaccharide repeating units containing two 2-N-acetyl-2-deoxy-d-galacturonic acids, and one of each of 2-N-acetyl-2-deoxy-d-glucose, d-glucose, d-ribose, and 7-N-acetylamino-3,5,7,9-tetradeoxy-5-N-[(R)-2-hydroxypropanoylamino]- l-glycero-l-manno-non-2-ulosonic acid. To our knowledge, this is the first finding a pseudaminic acid decorated with lactic acid residue in polysaccharides. The biological analysis showed that the capsular polysaccharide significantly reduced the viability and colony formation of HL-60 cells. Taken together, our data indicate that the capsular polysaccharide from P. marincola KMM 277T is a promising substance for the study of its antitumor properties and the mechanism of action in the future.

Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis.

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37-40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

Partial structure and immunological properties of lipopolysaccharide from marine-derived Pseudomonas stutzeri KMM 226.

The partial structure and immunology of the lipopolysaccharide (LPS) of Pseudomonas stutzeri KMM 226, a bacterium isolated from a seawater sample collected at a depth of 2000 m, was characterised. The O-polysaccharide was built up of disaccharide repeating units constituted by L-Rhap and D-GlcpNAc: →2)-α-L-Rhap-(1→3)-α-D-GlcpNAc-(1→. The structural analysis of the lipid A showed a mixture of different species. The major species were hexa-acylated and penta-acylated lipids A, bearing the 12:0(3-OH) in amide linkage and 10:0(3-OH) in ester linkage, while the secondary fatty acids were present only as 12:0. The presence of 12:0(2-OH) was not detected. The immunology experiments demonstrated that P. stutzeri KMM 226 LPS displayed a low ability to induce TNF-α, IL-1ß, IL-6, IL-8 and IL-10 cytokine production and acted as an antagonist of hexa-acylated Escherichia coli LPS in human blood in vitro.

Structure and in vitro antiproliferative activity against breast cancer cells of the cell-wall polysaccharide from the marine bacterium Kangiella japonica KMM 3899T.

Kangiella japonica KMM 3899T is a Gram-negative bacterium isolated from a sandy sediment sample collected from the Sea of Japan. Here the results of the structure and the biological activity against breast cancer cells of the cell-wall polysaccharide from K. japonica KMM 3899T have been described. The structure of the repeating unit of the polysaccharide was elucidated using chemical analysis and NMR spectroscopy: →4)-α-L-GalpNAc3AcA-(1 â†’ 3)-α-D-GlcpNAc-(1 â†’ 4)-ß-D-GlcpNAc3NAcAN-(1→. The cell-wall polysaccharide had an antiproliferative effect against T-47D cells. Flow cytometric and Western blot analysis revealed that the polysaccharide induced S phase arrest and mitochondrial-dependent apoptosis.

Lipopolysaccharides of Herbaspirillum species and their relevance for bacterium-host interactions.

The lipopolysaccharides of Herbaspirillum lusitanum P6-12T (HlP6-12T) and H. frisingense GSF30T (HfGSF30T) was isolated by phenol-water extraction from bacterial cells and was characterized using chemical analysis and SDS-PAGE. It was shown that these bacteria produce LPSs that differ in their physicochemical properties and macromolecular organization. In this paper, the lipid A structure of the HlP6-12T LPS, was characterized through chemical analyses and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. To prove the effect of the size of micelles on their bioavailability, we examined the activity of both LPSs toward the morphology of wheat seedlings. Analysis of the HlP6-12T and HfGSF30T genomes showed no significant differences between the operons that encode proteins involved in the biosynthesis of the lipids A and core oligosaccharides. The difference may be due to the composition of the O-antigen operon. HfGSF30T has two copies of the rfb operon, with the main one divided into two fragments. In contrast, the HlP6-12T genome contains only a single rfb-containing operon, and the other O-antigen operons are not comparable at all. The integrity of O-antigen-related genes may also affect LPS variability of. Specifically, we have observed a hairpin structure in the middle of the O-antigen glycosyltransferase gene, which led to the division of the gene into two fragments, resulting in incorrect protein synthesis and potential abnormalities in O-antigen production.

Structure of the O-polysaccharide from the moderately halophilic bacterium Halomonas fontilapidosi KR26.

Lipopolysaccharide was obtained from the aerobic moderately halophilic bacterium Halomonas fontilapidosi KR26. The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide and was examined by chemical methods and by 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC, and HMBC experiments. The following structure of the linear tetrasaccharide repeating unit was deduced. →2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-ß-d-Galp-(1→.

Cell-cycle arrest and mitochondria-dependent apoptosis induction in T-47D cells by the capsular polysaccharide from the marine bacterium Kangiella japonica KMM 3897.

In this study, we reported the in vitro mechanisms of antiproliferative activity of capsular polysaccharide derived from marine Gram-negative bacteria Kangiella japonica KMM 3897 in human breast сarcinoma T-47D cells. Flow cytometric and Western blot analysis revealed that capsular polysaccharide effectively suppressed T-47D cell proliferation by inducing G0/G1 phase arrest and mitochondrial-dependent apoptosis. Moreover, polysaccharide influenced the ERK1/2 and p38 signaling pathways. The results of this study would enrich our understanding of the molecular mechanism of the anti-cancer activity of sulfated polysaccharides from marine Gram-negative bacteria.

Sulfated capsular polysaccharide from the marine bacterium Kangiella japonica inhibits T-47D cells growth in vitro.

Kangiella japonica KMM 3897 is a Gram-negative bacterium isolated from a coastal sea-water sample of the Sea of Japan. In this paper, the results about the structure and the antiproliferative effect on cancer cells of the capsular polysaccharide isolated from the Kangiella japonica KMM 3897 have been described. The carbohydrate polymer was isolated and purified by several separation techniques, and the structure was elucidated using chemical analysis and NMR spectroscopy. The following structure of the sulfated capsular polysaccharide, containing 2-amino-2-deoxy-D-mannuronic acid was established: The capsular polysaccharide exerted a selective antiproliferative effect and suppressed the colony formation of T-47D cells.

Structure and antiproliferative activity of the polysaccharide from Halomonas aquamarina related to Cobetia pacifica.

Here, the results of the structure and the activity of capsular polysaccharides isolated from the Halomonas aquamarina EG27S8QL and Cobetia pacifica KMM3878 have been described. Both polysaccharides were studied by spectroscopic and chemical methods and were found to be structurally related sulfated galactans differing in the position of the sulfate group: →6)-ß-D-Galp3S-(1 â†’ 4)-ß-D-Galp3S-(1 â†’ 6)-ß-D-Galp3,4(S-Pyr)-(1 â†’ [H. aquamarina EG27S8QL] →6)-ß-D-Gal-(1 â†’ 4)-ß-D-Gal2,3S-(1 â†’ 6)-ß-D-Gal3,4(S-Pyr)-(1 â†’ [C. pacifica KMM3878] Structure of the CPS from H. aquamarina EG27S8QL has not been hitherto reported, whereas the CPS from C. pacifica KMM3878 was identical to the previously studied O-polysaccharide. The CPSs exhibited an antiproliferative effect and suppressed the colony formation of DLD-1 and MCF-7 cells in a different manner.

Structure and in vitro antiproliferative activity of the acidic capsular polysaccharide from the deep-sea bacterium Psychrobacter submarinus KMM 225T.

Psychrobacter submarinus KMM 225T is a Gram-negative bacterium isolated from a sea-water sample collected at a depth of 300 m in the Northwest Pacific Ocean. Here we report the structure of the capsular polysaccharide from P. submarinus KMM 225T and its effect on the viability and colony formation of cancer cells. The glycopolymer was purified by ultracentrifugation and chromatography methods, and the structure was elucidated using NMR spectroscopy and composition analyses. The following structure of the acidic capsular polysaccharide, containing 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido]-d-glucose [d-QuipNAc4N(S-Hb)] and 4,6-O-[(S)-1-carboxyethylidene]-2-acetamido-2-deoxy-d-glucose [d-GlcpNAc4,6(S-Pyr)] was established: The capsular polysaccharide slightly reduced the viability but effectively suppressed the colony formation of different types of cancer cells, of which the most pronounced inhibition was shown for the human chronic myelogenous leukemia K-562 cells.

The Unusual Lipid A Structure and Immunoinhibitory Activity of LPS from Marine Bacteria Echinicola pacifica KMM 6172T and Echinicola vietnamensis KMM 6221T.

Gram-negative bacteria experiencing marine habitats are constantly exposed to stressful conditions dictating their survival and proliferation. In response to these selective pressures, marine microorganisms adapt their membrane system to ensure protection and dynamicity in order to face the highly mutable sea environments. As an integral part of the Gram-negative outer membrane, structural modifications are commonly observed in the lipopolysaccharide (LPS) molecule; these mainly involve its glycolipid portion, i.e., the lipid A, mostly with regard to fatty acid content, to counterbalance the alterations caused by chemical and physical agents. As a consequence, unusual structural chemical features are frequently encountered in the lipid A of marine bacteria. By a combination of data attained from chemical, MALDI-TOF mass spectrometry (MS), and MS/MS analyses, here, we describe the structural characterization of the lipid A isolated from two marine bacteria of the Echinicola genus, i.e., E. pacifica KMM 6172T and E. vietnamensis KMM 6221T. This study showed for both strains a complex blend of mono-phosphorylated tri- and tetra-acylated lipid A species carrying an additional sugar moiety, a d-galacturonic acid, on the glucosamine backbone. The unusual chemical structures are reflected in a molecule that only scantly activates the immune response upon its binding to the LPS innate immunity receptor, the TLR4-MD-2 complex. Strikingly, both LPS potently inhibited the toxic effects of proinflammatory Salmonella LPS on human TLR4/MD-2.

Immunomodulatory Properties of Polysaccharides from the Coral Pseudopterogorgia americana in Macrophages.

Polysaccharides from marine organisms produce an important regulatory effect on the mammalian immune system. In this study, the immunomodulatory properties of a polysaccharide that was isolated from the coral Pseudopterogorgia americana (PPA) were investigated. PPA increased the expression levels of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2), but not inducible nitric oxide synthase and nitric oxide, in macrophages. A mechanistic study revealed that PPA activated macrophages through the toll-like receptor-4 and induced the generation of reactive oxygen species (ROS), increased the phosphorylation levels of protein kinase C (PKC)-α, PKC-δ and mitogen-activated protein kinases (MAPK), and activated NF-κB. The inhibition of ROS and knockdown of PKC-α reduced PPA-mediated TNF-α and IL-6 expression; however, the knockdown of PKC-δ significantly increased PPA-mediated TNF-α expression. In addition, the inhibition of c-Jun N-terminal kinase-1/2 and NF-κB reduced PPA-mediated TNF-α, IL-6 and COX-2 expression. Furthermore, the inhibition of ROS, MAPK and PKC-α/δ reduced PPA-mediated NF-κB activation, indicating that ROS, MAPK and PKC-α/δ function as upstream signals of NF-κB. Finally, PPA treatment decreased the phagocytosis activity of macrophages and reduced cytokine expression in bacteria-infected macrophages. Taken together, our current findings suggest that PPA can potentially play a role in the development of immune modulators in the future.