Publications by authors named "Rosa Lanzetta"

136 Publications

Physicochemical Approach to Understanding the Structure, Conformation, and Activity of Mannan Polysaccharides.

Biomacromolecules 2021 Apr 17;22(4):1445-1457. Epub 2021 Mar 17.

Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Naples, Italy.

Extracellular polysaccharides are widely produced by bacteria, yeasts, and algae. These polymers are involved in several biological functions, such as bacteria adhesion to surface and biofilm formation, ion sequestering, protection from desiccation, and cryoprotection. The chemical characterization of these polymers is the starting point for obtaining relationships between their structures and their various functions. While this fundamental correlation is well reported and studied for the proteins, for the polysaccharides, this relationship is less intuitive. In this paper, we elucidate the chemical structure and conformational studies of a mannan exopolysaccharide from the permafrost isolated bacterium strain 273-4. The mannan from the cold-adapted bacterium was compared with its dephosphorylated derivative and the commercial product from . Starting from the chemical structure, we explored a new approach to deepen the study of the structure/activity relationship. A pool of physicochemical techniques, ranging from small-angle neutron scattering (SANS) and dynamic and static light scattering (DLS and SLS, respectively) to circular dichroism (CD) and cryo-transmission electron microscopy (cryo-TEM), have been used. Finally, the ice recrystallization inhibition activity of the polysaccharides was explored. The experimental evidence suggests that the mannan exopolysaccharide from bacterium has an efficient interaction with the water molecules, and it is structurally characterized by rigid-rod regions assuming a 14-helix-type conformation.
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http://dx.doi.org/10.1021/acs.biomac.0c01659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8045027PMC
April 2021

Levan from a new isolated Bacillus subtilis AF17: Purification, structural analysis and antioxidant activities.

Int J Biol Macromol 2020 Feb 15;144:316-324. Epub 2019 Dec 15.

Laboratory for the Improvement of Plants and Valorization of Agroressources, National School of Engineering of Sfax (ENIS), Soukra Road, km 3.5, B.P. 1173, 3038 Sfax, Tunisia; Common Service Unit of Bioreactor Coupled with an Ultrafilter, National School of Engineering, Sfax University, P.O. Box 1173, 3038 Sfax, Tunisia.

A strain of Bacillus subtilis AF 17 with high exopolysaccharide (EPS) production ability was isolated and identified based on morphological and physiological characteristics and phylogenetic analysis of 16S rDNA sequences. EPS was isolated from the strain fermentation broth by alcohol precipitation and gel-filtration chromatography. Its structural characteristics were investigated and elucidated by methylation analysis, gas chromatography mass spectrometry and nuclear magnetic resonance spectroscopy. Based on the obtained data, the EPS was found to be a levan containing a backbone of 6-substituted β-fructoses, with a low grade of branching at position 1 (linear/branched ratio 20:1). Levan showed a molecular weight of about 20 MDa. The antioxidant activity of this biopolymer was studied and revealed that levan showed an interesting 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity (IC = 1.42 mg/mL), reducing power, and also a strong total antioxidant activity. Overall, the results suggest that levan is a promising source of natural antioxidants and can be used as additive in food and pharmaceutical preparations.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.12.108DOI Listing
February 2020

Characterisation of the Dynamic Interactions between Complex N-Glycans and Human CD22.

Chembiochem 2020 01 17;21(1-2):129-140. Epub 2019 Oct 17.

Dipartimento di Scienze Chimiche, Complesso Universitario Monte Sant'Angelo, Università di Napoli Federico II, Via Cintia 4, 80126, Napoli, Italy.

CD22 (Siglec-2) is a B-cell surface inhibitory protein capable of selectively recognising sialylated glycans, thus dampening autoimmune responses against self-antigens. Here we have characterised the dynamic recognition of complex-type N-glycans by human CD22 by means of orthogonal approaches including NMR spectroscopy, computational methods and biophysical assays. We provide new molecular insights into the binding mode of sialoglycans in complex with h-CD22, highlighting the role of the sialic acid galactose moieties in the recognition process, elucidating the conformational behaviour of complex-type N-glycans bound to Siglec-2 and dissecting the formation of CD22 homo-oligomers on the B-cell surface. Our results could enable the development of additional therapeutics capable of modulating the activity of h-CD22 in autoimmune diseases and malignancies derived from B-cells.
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http://dx.doi.org/10.1002/cbic.201900295DOI Listing
January 2020

Isolation and structural characterization of levan produced by probiotic Bacillus tequilensis-GM from Tunisian fermented goat milk.

Int J Biol Macromol 2019 Jul 17;133:786-794. Epub 2019 Apr 17.

Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Naples, Italy.

The probiotic features of strain GM newly isolated from Tunisian spontaneously fermented goat milk and identified as Bacillus tequilensis-GM were assessed. Strain GM showed high resistance to saliva (90.64%), gastric juice (88.55%), intestinal juice (72.83%) and resistance to bile salts (65.22%), was able to act against Listeria monocytogenes ATCC 15313, Escherichia coli ATCC 25922 and Enterococcus feacalis ATCC 25912, showed high surface hydrophobicity (77.3%) and was sensitive to most of the studied antibiotics. Strain GM did not exhibit any hemolytic activity whereas it was able to produce protease, amylase and β-galactosidase. Moreover, results showed that strain GM produced high molecular weight β-(2 → 6)-levan with high ability to inhibit and to disrupt pathogenic biofilms and with high ability to reduce syneresis of sucrose-supplemented skimmed milk. B. tequilensis-GM can therefore be suitable to be used as starter culture in fermented dairy products, since it possesses desirable probiotic properties in addition to its ability to produce levan.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.04.130DOI Listing
July 2019

Human Macrophage Galactose-Type Lectin (MGL) Recognizes the Outer Core of Escherichia coli Lipooligosaccharide.

Chembiochem 2019 07 24;20(14):1778-1782. Epub 2019 Jun 24.

Department of Chemical Sciences, University of Naples Federico II, via Cintia 4, 80126, Napoli, Italy.

Carbohydrate-lectin interactions intervene in and mediate most biological processes, including a crucial modulation of immune responses to pathogens. Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. Herein, a novel molecular interaction between the human macrophage galactose-type lectin (MGL) and the lipooligosaccharide (LOS) of Escherichia coli strain R1 is described. Saturation transfer difference NMR spectroscopy analysis, supported by computational studies, demonstrated that MGL bound to the purified deacylated LOS mainly through recognition of its outer core and established crucial interactions with the terminal Galα(1,2)Gal epitope. These results assess the ability of MGL to recognise glycan moieties exposed on Gram-negative bacterial surfaces.
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http://dx.doi.org/10.1002/cbic.201900087DOI Listing
July 2019

Lipid A: Immunological Properties and Molecular Basis of Its Binding to the Myeloid Differentiation Protein-2/Toll-Like Receptor 4 Complex.

Front Immunol 2018 14;9:1888. Epub 2018 Aug 14.

Dipartimento di Scienze Chimiche, Complesso Universitario Monte Sant'Angelo, Università di Napoli Federico II, Naples, Italy.

Lipopolysaccharides (LPS) are potent activator of the innate immune response through the binding to the myeloid differentiation protein-2 (MD-2)/toll-like receptor 4 (TLR4) receptor complexes. Although a variety of LPSs have been characterized so far, a detailed molecular description of the structure-activity relationship of the lipid A part has yet to be clarified. Photosynthetic strains, symbiont of legumes, express distinctive LPSs bearing very long-chain fatty acids with a hopanoid moiety covalently linked to the lipid A region. Here, we investigated the immunological properties of LPSs isolated from strains on both murine and human immune systems. We found that they exhibit a weak agonistic activity and, more interestingly, a potent inhibitory effect on MD-2/TLR4 activation exerted by toxic enterobacterial LPSs. By applying computational modeling techniques, we also furnished a plausible explanation for the LPS inhibitory activity at atomic level, revealing that its uncommon lipid A chemical features could impair the proper formation of the receptorial complex, and/or has a destabilizing effect on the pre-assembled complex itself.
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http://dx.doi.org/10.3389/fimmu.2018.01888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102379PMC
September 2019

Lipid A structural characterization from the LPS of the Siberian psychro-tolerant Psychrobacter arcticus 273-4 grown at low temperature.

Extremophiles 2018 Nov 20;22(6):955-963. Epub 2018 Aug 20.

Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Naples, Italy.

Psychrobacter arcticus 273-4 is a Gram-negative bacterium isolated from a 20,000-to-30,000-year-old continuously frozen permafrost in the Kolyma region in Siberia. The survival strategies adopted to live at subzero temperatures include all the outer membrane molecules. A strategic involvement in the well-known enhancement of cellular membrane fluidity is attributable to the lipopolysaccharides (LPSs). These molecules covering about the 75% of cellular surface contribute to cold adaptation through structural modifications in their portions. In this work, we elucidated the exact structure of lipid A moiety obtained from the lipopolysaccharide of P. arcticus grown at 4 °C, to mimic the response to the real environment temperatures. The lipid A was obtained from the LPS by mild acid hydrolysis. The lipid A and its partially deacylated derivatives were exhaustively characterized by chemical analysis and by means of ESI Q-Orbitrap mass spectrometry. Moreover, biological assays indicated that P. arcticus 273-4 lipid A may behave as a weak TLR4 agonist.
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http://dx.doi.org/10.1007/s00792-018-1051-6DOI Listing
November 2018

Rhodopseudomonas palustris Strain CGA009 Produces an O-Antigen Built up by a C-4-Branched Monosaccharide: Structural and Conformational Studies.

Org Lett 2018 06 6;20(12):3656-3660. Epub 2018 Jun 6.

Dipartimento di Scienze Chimiche , Complesso Universitario Monte Sant'Angelo, Università di Napoli Federico II , Via Cintia 4 , I-80126 Napoli , Italy.

Here, the analysis of the peculiar homopolymeric O-chain, isolated from the lipopolysaccharide (LPS) of Rhodopseudomonas palustris strain CGA009, is reported. The O-chain is built up of a novel 4-C-branched sugar (12-deoxy-4- C-(d- altro-5,7,8,9-tetrahydroxyhexyl))-3- O-methyl-d-galactopyranose)) whose structure, absolute configuration, and conformational features were deduced by 2D NMR spectroscopy, optical rotation measurements, and molecular dynamics simulations.
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http://dx.doi.org/10.1021/acs.orglett.8b01439DOI Listing
June 2018

The Structure of the Lipid A from the Halophilic Bacterium Spiribacter salinus M19-40.

Mar Drugs 2018 Apr 11;16(4). Epub 2018 Apr 11.

Department of Chemical Sciences, University of Naples Federico II, 80126 Naples, Italy.

The study of the adaptation mechanisms that allow microorganisms to live and proliferate in an extreme habitat is a growing research field. Directly exposed to the external environment, lipopolysaccharides (LPS) from Gram-negative bacteria are of great appeal as they can present particular structural features that may aid the understanding of the adaptation processes. Moreover, through being involved in modulating the mammalian immune system response in a structure-dependent fashion, the elucidation of the LPS structure can also be seen as a fundamental step from a biomedical point of view. In this paper, the lipid A structure of the LPS from M19-40, a halophilic gamma-proteobacteria, was characterized through chemical analyses and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This revealed a mixture of mono- and bisphosphorylated penta- to tri-acylated species with the uncommon 2 + 3 symmetry and bearing an unusual 3-oxotetradecaonic acid.
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http://dx.doi.org/10.3390/md16040124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923411PMC
April 2018

Exopolysaccharides from Marine and Marine Extremophilic Bacteria: Structures, Properties, Ecological Roles and Applications.

Mar Drugs 2018 Feb 20;16(2). Epub 2018 Feb 20.

Department of Chemical Sciences, University of Naples "Federico II", Naples 80126, Italy.

The marine environment is the largest aquatic ecosystem on Earth and it harbours microorganisms responsible for more than 50% of total biomass of prokaryotes in the world. All these microorganisms produce extracellular polymers that constitute a substantial part of the dissolved organic carbon, often in the form of exopolysaccharides (EPS). In addition, the production of these polymers is often correlated to the establishment of the biofilm growth mode, during which they are important matrix components. Their functions include adhesion and colonization of surfaces, protection of the bacterial cells and support for biochemical interactions between the bacteria and the surrounding environment. The aim of this review is to present a summary of the status of the research about the structures of exopolysaccharides from marine bacteria, including capsular, medium released and biofilm embedded polysaccharides. Moreover, ecological roles of these polymers, especially for those isolated from extreme ecological niches (deep-sea hydrothermal vents, polar regions, hypersaline ponds, etc.), are reported. Finally, relationships between the structure and the function of the exopolysaccharides are discussed.
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http://dx.doi.org/10.3390/md16020069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852497PMC
February 2018

Structure of the chlorovirus PBCV-1 major capsid glycoprotein determined by combining crystallographic and carbohydrate molecular modeling approaches.

Proc Natl Acad Sci U S A 2018 01 18;115(1):E44-E52. Epub 2017 Dec 18.

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-2032;

The glycans of the major capsid protein (Vp54) of chlorella virus (PBCV-1) were recently described and found to be unusual. This prompted a reexamination of the previously reported Vp54 X-ray structure. A detailed description of the complete glycoprotein was achieved by combining crystallographic data with molecular modeling. The crystallographic data identified most of the monosaccharides located close to the protein backbone, but failed to detect those further from the glycosylation sites. Molecular modeling complemented this model by adding the missing monosaccharides and examined the conformational preference of the whole molecule, alone or within the crystallographic environment. Thus, combining X-ray crystallography with carbohydrate molecular modeling resulted in determining the complete glycosylated structure of a glycoprotein. In this case, it is the chlorovirus PBCV-1 major capsid protein.
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http://dx.doi.org/10.1073/pnas.1613432115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5776783PMC
January 2018

Protective effect of Opuntia ficus-indica L. cladodes against UVA-induced oxidative stress in normal human keratinocytes.

Bioorg Med Chem Lett 2017 12 20;27(24):5485-5489. Epub 2017 Oct 20.

Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte Sant'Angelo, via Cinthia 4, 80126 Naples, Italy. Electronic address:

Opuntia ficus-indica L. is known for its beneficial effects on human health, but still little is known on cladodes as a potent source of antioxidants. Here, a direct, economic and safe method was set up to obtain water extracts from Opuntia ficus-indica cladodes rich in antioxidant compounds. When human keratinocytes were pre-treated with the extract before being exposed to UVA radiations, a clear protective effect against UVA-induced stress was evidenced, as indicated by the inhibition of stress-induced processes, such as free radicals production, lipid peroxidation and GSH depletion. Moreover, a clear protective effect against apoptosis in pre-treated irradiated cells was evidenced. We found that eucomic and piscidic acids were responsible for the anti-oxidative stress action of cladode extract. In conclusion, a bioactive, safe, low-cost and high value-added extract from Opuntia cladodes was obtained to be used for skin health/protection.
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http://dx.doi.org/10.1016/j.bmcl.2017.10.043DOI Listing
December 2017

Production and structural characterization of exopolysaccharides from newly isolated probiotic lactic acid bacteria.

Int J Biol Macromol 2018 Mar 31;108:719-728. Epub 2017 Oct 31.

Université de Sfax, ENIS, Laboratoire Analyse, Valorisation et Sécurité des Aliments, Sfax, 3038, Tunisia. Electronic address:

In this work, four exopolysaccharide-producing lactic acid bacteria (LAB) strains, newly isolated from Tunisian spontaneously fermented foods and beverages, namely bovine and turkey meat sausages (BMS and TMS), date palm sap (DPS) and cow milk (CM), were identified as Leuconostoc citreum-BMS, Leuconostoc mesenteroides-TMS, Pediococcus pentosaceus-DPS and Leuconostoc pseudomesenteroides-CM, respectively. The isolated strains showed the ability to withstand simulated human gastrointestinal (GI) tract conditions (low pH, lysozyme, bile salts, pepsin and pancreatin) and showed high surface hydrophobicity (79-90%), besides their ability to act against Escherichia coli and Listeria monocytogenes and to produce exopolysaccharides (EPS). Therefore, these isolates can be served as potential probiotics. The produced EPS were growth-associated suggesting that they are primary metabolites. The molecular weights were higher than 10Da using HPLC-SEC. 2D-NMR results indicated that all the samples were mixtures of dextran and levan, except for EPS-CM which was a levan-type EPS. Furthermore, the EPS samples showed an abitlity to inhibit and to disrupt pathogenic biofilms and showed high thermostability studied via differential scanning calorimetry (DSC) with melting points higher than 224°C making them promising to be used in thermal processed foods.
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http://dx.doi.org/10.1016/j.ijbiomac.2017.10.155DOI Listing
March 2018

Unusual Lipid A from a Cold-Adapted Bacterium: Detailed Structural Characterization.

Chembiochem 2017 09 27;18(18):1845-1854. Epub 2017 Jul 27.

Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Naples, Italy.

Colwellia psychrerythraea 34H is a Gram-negative cold-adapted microorganism that adopts many strategies to cope with the limitations associated with the low temperatures of its habitat. In this study, we report the complete characterization of the lipid A moiety from the lipopolysaccharide of Colwellia. Lipid A and its partially deacylated derivative were completely characterized by high-resolution mass spectrometry, NMR spectroscopy, and chemical analysis. An unusual structure with a 3-hydroxy unsaturated tetradecenoic acid as a component of the primary acylation pattern was identified. In addition, the presence of a partially acylated phosphoglycerol moiety on the secondary acylation site at the 3-position of the reducing 2-amino-2-deoxyglucopyranose unit caused tremendous natural heterogeneity in the structure of lipid A. Biological-activity assays indicated that C. psychrerythraea 34H lipid A did not show an agonistic or antagonistic effect upon testing in human macrophages.
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http://dx.doi.org/10.1002/cbic.201700287DOI Listing
September 2017

Xanthomonas citri pv. citri Pathotypes: LPS Structure and Function as Microbe-Associated Molecular Patterns.

Chembiochem 2017 04 20;18(8):772-781. Epub 2017 Mar 20.

Department of Chemical Sciences, University of Naples Federico II, via Cinthia 4, 80126, Naples, Italy.

Xanthomonas citri pv. citri is the pathogen responsible for Asiatic citrus canker, one of the most serious citrus diseases worldwide. The lipopolysaccharide (LPS) molecule has been demonstrated to be involved in X. citri pv. citri virulence. Despite enormous progress in investigations of the molecular mechanisms for bacterial pathogenicity, determination of the detailed LPS structure-activity relationship is limited, as the current knowledge is mainly based on structural determination of one X. citri pv. citri strain. As X. citri pv. citri strains are distinguished into three main pathogenicity groups, we characterized the full structure of the LPS from two pathotypes that differ in their host-range specificity. This revealed an intriguing difference in LPS O-chain structure. We also tested the LPSs and isolated lipid A moieties for their ability to act as microbe-associated molecular patterns in Arabidopsis thaliana. Both LPS/lipid As induced ROS accumulation, but no difference was observed between the two pathotypes.
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http://dx.doi.org/10.1002/cbic.201600671DOI Listing
April 2017

Structural characterization of an all-aminosugar-containing capsular polysaccharide from Colwellia psychrerythraea 34H.

Antonie Van Leeuwenhoek 2017 Nov 4;110(11):1377-1387. Epub 2017 Feb 4.

Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Naples, Italy.

Colwellia psychrerythraea strain 34H, a Gram-negative bacterium isolated from Arctic marine sediments, is considered a model to study the adaptation to cold environments. Recently, we demonstrated that C. psychrerythraea 34H produces two different extracellular polysaccharides, a capsular polysaccharide and a medium released polysaccharide, which confer cryoprotection to the bacterium. In this study, we report the structure of an additional capsular polysaccharide produced by Colwellia grown at a different temperature. The structure was determined using chemical methods, and one- and two-dimensional NMR spectroscopy. The results showed a trisaccharide repeating unit made up of only amino-sugar residues: N-acetyl-galactosamine, 2,4-diacetamido-2,4,6-trideoxy-glucose (bacillosamine), and 2-acetamido-2-deoxyglucuronic acid with the following structure: →4)-β-D-GlcpNAcA-(1 →3)-β-D-QuipNAc4NAc-(1 →3)-β-D-GalpNAc-(1 →. The 3D model, generated in accordance with H,H-NOE NMR correlations and consisting of ten repeating units, shows a helical structure. In contrast with the other extracellular polysaccharides produced from Colwellia at 4 °C, this molecule displays only a low ice recrystallization inhibition activity.
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http://dx.doi.org/10.1007/s10482-017-0834-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5718298PMC
November 2017

The Lipid A from Rhodopseudomonas palustris Strain BisA53 LPS Possesses a Unique Structure and Low Immunostimulant Properties.

Chemistry 2017 Mar 22;23(15):3637-3647. Epub 2016 Dec 22.

Department of Chemical Sciences, University of Naples, Federico II, via Cinthia 4, 80126, Naples, Italy.

The search for novel lipid A analogues from any biological source that can act as antagonists, displaying inhibitory activity towards the production of pro-inflammatory cytokines, or as immunomodulators in mammals, is a very topical issue. To this aim, the structure and immunological properties of the lipopolysaccharide lipid A from the purple nonsulfur bacterium Rhodopseudomonas palustris strain BisA53 have been determined. This lipid A displays a unique structural feature, with a non-phosphorylated skeleton made up of the tetrasaccharide Manp-α-(1→4)-GlcpN3N-β-1→6-GlcpN3N-α-(1→1)-α-GalpA, and four primary amide-linked 14:0(3-OH) and, as secondary O-acyl substituents, a 16:0 and the very long-chain fatty acid 26:0(25-OAc), appended on the GlcpN3N units. This lipid A architecture is definitely rare, so far identified only in the genus Bradyrhizobium. Immunological tests on both murine bone-marrow-derived and human monocyte-derived macrophages revealed an extremely low immunostimulant capability of this LPS lipid A.
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http://dx.doi.org/10.1002/chem.201604379DOI Listing
March 2017

The polysaccharide and low molecular weight components of Opuntia ficus indica cladodes: Structure and skin repairing properties.

Carbohydr Polym 2017 Feb 24;157:128-136. Epub 2016 Sep 24.

Department of Chemical Sciences, University of Naples Federico II, Via Cintia 4, I-80126 Napoli, Italy. Electronic address:

The Opuntia ficus-indica multiple properties are reflected in the increasing interest of chemists in the identification of its natural components having pharmaceutical and/or cosmetical applications. Here we report the structural elucidation of Opuntia ficus-indica mucilage that highlighted the presence of components differing for their chemical nature and the molecular weight distribution. The high molecular weight components were identified as a linear galactan polymer and a highly branched xyloarabinan. The low molecular weight components were identified as lactic acid, D-mannitol, piscidic, eucomic and 2-hydroxy-4-(4'-hydroxyphenyl)-butanoic acids. A wound healing assay was performed in order to test the cicatrizing properties of the various components, highlighting the ability of these latter to fasten dermal regeneration using a simplified in vitro cellular model based on a scratched keratinocytes monolayer. The results showed that the whole Opuntia mucilage and the low molecular weight components are active in the wound repair.
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http://dx.doi.org/10.1016/j.carbpol.2016.09.073DOI Listing
February 2017

Structure-activity relationship of the exopolysaccharide from a psychrophilic bacterium: A strategy for cryoprotection.

Carbohydr Polym 2017 Jan 14;156:364-371. Epub 2016 Sep 14.

Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Naples, Italy. Electronic address:

Microrganisms from sea ice, glacial and subglacial environments are currently under investigation due to their relevant ecological functions in these habitats, and to their potential biotechnological applications. The cold-adapted Colwellia psychrerythraea 34H produces extracellular polysaccharides with cryoprotection activity. We here describe the purification and detailed molecular primary and secondary structure of the exopolysaccharide (EPS) secreted by C. psychrerythraea 34H cells grown at 4°C. The structure was determined by chemical analysis and NMR. The trisaccharide repeating unit of the EPS is constituted by a N-acetyl quinovosamine unit and two residues of galacturonic acid both decorated with alanine. In addition, the EPS was tested in vitro showing a significant inhibitory effect on ice recrystallization. In-depth NMR and computational analysis suggest a pseudohelicoidal structure which seems to prevent the local tetrahedral order of the water molecules in the first hydration shell, and could be responsible of the inhibition of ice recrystallization. As cell cryopreservation is an essential tool in modern biotechnology and medicine, the observations reported in this paper could pave the way for a biotechnological application of Colwellia EPS.
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http://dx.doi.org/10.1016/j.carbpol.2016.09.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5166977PMC
January 2017

Structural characterization of the lipid A from the LPS of the haloalkaliphilic bacterium Halomonas pantelleriensis.

Extremophiles 2016 Sep 21;20(5):687-94. Epub 2016 Jun 21.

Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Naples, Italy.

Halomonas pantelleriensis DSM9661(Τ) is a Gram-negative haloalkaliphilic bacterium isolated from the sand of the volcanic Venus mirror lake, closed to seashore in the Pantelleria Island in the south of Italy. It is able to optimally grow in media containing 3-15 % (w/v) total salt and at pH between 9 and 10. To survive in these harsh conditions, the bacterium has developed several strategies that probably concern the bacteria outer membrane, a barrier regulating the exchange with the environment. In such a context, the lipopolysaccharides (LPSs), which are among the major constituent of the Gram-negative outer membrane, are thought to contribute to the restrictive membrane permeability properties. The structure of the lipid A family derived from the LPS of Halomonas pantelleriensis DSM 9661(T) is reported herein. The lipid A was obtained from the purified LPS by mild acid hydrolysis. The lipid A, which contains different numbers of fatty acids residues, and its partially deacylated derivatives were completely characterized by means of ESI FT-ICR mass spectrometry and chemical analysis. Preliminary immunological assays were performed, and a comparison with the lipid A structure of the phylogenetic proximal Halomonas magadiensis is also reported.
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http://dx.doi.org/10.1007/s00792-016-0858-2DOI Listing
September 2016

The structure of the lipooligosaccharide from Xanthomonas oryzae pv. Oryzae: the causal agent of the bacterial leaf blight in rice.

Carbohydr Res 2016 Jun 4;427:38-43. Epub 2016 Apr 4.

Department of Chemical Sciences, University of Naples Federico II, via Cinthia 4, 80126 Naples, Italy. Electronic address:

The structure of the lipooligosaccharide (LOS) from the rice pathogen Xanthomonas oryzae pv. oryzae has been elucidated. The characterization of the core oligosaccharide structure was obtained by the employment of two chemical degradation protocols and by analysis of the products via NMR spectroscopy. The structure of the lipid A portion was achieved by MALDI mass spectrometry analysis on purified lipid A. The LOS from Xanthomonas oryzae pv. oryzae revealed to possess the same core structure of Xanthomonas campestris pv. campestris and interesting novel features on its lipid A domain. The evaluation of the biological activity of both LOS and isolated lipid A was also executed.
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http://dx.doi.org/10.1016/j.carres.2016.03.026DOI Listing
June 2016

N-Linked Glycans of Chloroviruses Sharing a Core Architecture without Precedent.

Angew Chem Int Ed Engl 2016 Jan 19;55(2):654-8. Epub 2015 Nov 19.

Department of Plant Pathology and Nebraska Center for Virology, University of Nebraska, Lincoln, NE 68583-0900 (USA).

N-glycosylation is a fundamental modification of proteins and exists in the three domains of life and in some viruses, including the chloroviruses, for which a new type of core N-glycan is herein described. This N-glycan core structure, common to all chloroviruses, is a pentasaccharide with a β-glucose linked to an asparagine residue which is not located in the typical sequon N-X-T/S. The glucose is linked to a terminal xylose unit and a hyperbranched fucose, which is in turn substituted with a terminal galactose and a second xylose residue. The third position of the fucose unit is always linked to a rhamnose, which is a semiconserved element because its absolute configuration is virus-dependent. Additional decorations occur on this core N-glycan and represent a molecular signature for each chlorovirus.
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http://dx.doi.org/10.1002/anie.201509150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836869PMC
January 2016

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

Mar Drugs 2015 Jul 22;13(7):4539-55. Epub 2015 Jul 22.

Dipartimento di Scienze Chimiche, Università degli Studi di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, Napoli 80126, Italy.

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 (1)H and (13)C 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.
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http://dx.doi.org/10.3390/md13074539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4515632PMC
July 2015

Burkholderia pseudomallei Capsular Polysaccharide Recognition by a Monoclonal Antibody Reveals Key Details toward a Biodefense Vaccine and Diagnostics against Melioidosis.

ACS Chem Biol 2015 Oct 5;10(10):2295-302. Epub 2015 Aug 5.

Department of Chemical Sciences, Università di Napoli Federico II , Complesso Universitario Monte S. Angelo, Via Cintia 4, I-80126, Naples, Italy.

Burkholderia pseudomallei is the bacterium responsible for melioidosis, an infectious disease with high mortality rates. Since melioidosis is a significant public health concern in endemic regions and the organism is currently classified as a potential biothreat agent, the development of effective vaccines and rapid diagnostics is a priority. The capsular polysaccharide (CPS) expressed by B. pseudomallei is a highly conserved virulence factor and a protective antigen. Because of this, CPS is considered an attractive antigen for use in the development of both vaccines and diagnostics. In the present study, we describe the interactions of CPS with the murine monoclonal antibody (mAb) 4C4 using a multidisciplinary approach including organic synthesis, molecular biology techniques, surface plasmon resonance, and nuclear magnetic spectroscopy. Using these methods, we determined the mode of binding between mAb 4C4 and native CPS or ad hoc synthesized capsular polysaccharide fragments. Interestingly, we demonstrated that the O-acetyl moiety of CPS is essential for the interaction of the CPS epitope with mAb 4C4. Collectively, our results provide important insights into the structural features of B. pseudomallei CPS that enable antibody recognition that may help the rational design of CPS-based vaccine candidates. In addition, our findings confirm that the mAb 4C4 is suitable for use in an antibody-based detection assay for diagnosis of B. pseudomallei infections.
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http://dx.doi.org/10.1021/acschembio.5b00502DOI Listing
October 2015

Activation of Human Toll-like Receptor 4 (TLR4)·Myeloid Differentiation Factor 2 (MD-2) by Hypoacylated Lipopolysaccharide from a Clinical Isolate of Burkholderia cenocepacia.

J Biol Chem 2015 Aug 9;290(35):21305-19. Epub 2015 Jul 9.

From the Departments of Chemical Sciences and

Lung infection by Burkholderia species, in particular Burkholderia cenocepacia, accelerates tissue damage and increases post-lung transplant mortality in cystic fibrosis patients. Host-microbe interplay largely depends on interactions between pathogen-specific molecules and innate immune receptors such as Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4·myeloid differentiation factor 2 (MD-2) LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4·MD-2 despite its lipid A having only five acyl chains. Furthermore, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the proinflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling combined with mutagenesis of TLR4-MD-2 interactive surfaces suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4·MD-2 complex by penta-acylated lipid A explaining the ability of hypoacylated B. cenocepacia LPS to promote proinflammatory responses associated with the severe pathogenicity of this opportunistic bacterium.
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http://dx.doi.org/10.1074/jbc.M115.649087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571861PMC
August 2015

Determination of the structure of the O-antigen and the lipid A from the entomopathogenic bacterium Pseudomonas entomophila lipopolysaccharide along with its immunological properties.

Carbohydr Res 2015 Aug 1;412:20-7. Epub 2015 May 1.

Dipartimento di Agraria, Via Università 100, 80055 Portici, NA, Italy. Electronic address:

The structure and the immunology of the lipopolysaccharide (LPS) of Pseudomonas entomophila, an entomopathogenic bacterium isolated from the fruit fly Drosophila melanogaster, was characterized. The O-antigen portion was established and resulted to be built up of a repetitive unit constituted by four monosaccharide residues, all L configured, all deoxy at C-6 and with an acetamido function at C-2: →3)-α-l-FucNAc-(1→4)-α-l-FucNAc-(1→3)-α-l-FucNAc-(1→3)-β-l-QuiNAc-(1→ The structural analysis of lipid A, showed a mixture of different species. The diphosphorylated glucosamine backbone carries six fatty acids consistent with the composition C10:0 3(OH), C12:0 2(OH) and C12:0 3(OH), whereas other species differs by the number of phosphates and/or of fatty acids. The immunology experiments demonstrated that the LPS structure of P. entomophila displayed a low ability to engage the TLR4-mediated signaling correlated to a significant antagonistic activity toward hexa-acylated LPS structures.
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http://dx.doi.org/10.1016/j.carres.2015.04.017DOI Listing
August 2015

A unique capsular polysaccharide structure from the psychrophilic marine bacterium Colwellia psychrerythraea 34H that mimics antifreeze (glyco)proteins.

J Am Chem Soc 2015 Jan 2;137(1):179-89. Epub 2015 Jan 2.

Department of Chemical Sciences, University of Naples Federico II , Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Naples, Italy.

The low temperatures of polar regions and high-altitude environments, especially icy habitats, present challenges for many microorganisms. Their ability to live under subfreezing conditions implies the production of compounds conferring cryotolerance. Colwellia psychrerythraea 34H, a γ-proteobacterium isolated from subzero Arctic marine sediments, provides a model for the study of life in cold environments. We report here the identification and detailed molecular primary and secondary structures of capsular polysaccharide from C. psychrerythraea 34H cells. The polymer was isolated in the water layer when cells were extracted by phenol/water and characterized by one- and two-dimensional NMR spectroscopy together with chemical analysis. Molecular mechanics and dynamics calculations were also performed. The polysaccharide consists of a tetrasaccharidic repeating unit containing two amino sugars and two uronic acids bearing threonine as substituent. The structural features of this unique polysaccharide resemble those present in antifreeze proteins and glycoproteins. These results suggest a possible correlation between the capsule structure and the ability of C. psychrerythraea to colonize subfreezing marine environments.
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http://dx.doi.org/10.1021/ja5075954DOI Listing
January 2015

Covalently linked hopanoid-lipid A improves outer-membrane resistance of a Bradyrhizobium symbiont of legumes.

Nat Commun 2014 Oct 30;5:5106. Epub 2014 Oct 30.

Dipartimento di Scienze Chimiche, Complesso Universitario Monte Sant'Angelo, Università di Napoli Federico II, Via Cintia 4, Napoli I-80126, Italy.

Lipopolysaccharides (LPSs) are major components of the outer membrane of Gram-negative bacteria and are essential for their growth and survival. They act as a structural barrier and play an important role in the interaction with eukaryotic hosts. Here we demonstrate that a photosynthetic Bradyrhizobium strain, symbiont of Aeschynomene legumes, synthesizes a unique LPS bearing a hopanoid covalently attached to lipid A. Biophysical analyses of reconstituted liposomes indicate that this hopanoid-lipid A structure reinforces the stability and rigidity of the outer membrane. In addition, the bacterium produces other hopanoid molecules not linked to LPS. A hopanoid-deficient strain, lacking a squalene hopene cyclase, displays increased sensitivity to stressful conditions and reduced ability to survive intracellularly in the host plant. This unusual combination of hopanoid and LPS molecules may represent an adaptation to optimize bacterial survival in both free-living and symbiotic states.
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http://dx.doi.org/10.1038/ncomms6106DOI Listing
October 2014

Structural and conformational study of the O-polysaccharide produced by the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris strain BisA53.

Carbohydr Polym 2014 Dec 27;114:384-391. Epub 2014 Aug 27.

Dipartimento di Scienze Chimiche, Complesso Universitario Monte Sant'Angelo, Università di Napoli Federico II, Via Cintia 4, I-80126 Napoli, Italy.

Rhodopseudomonas palustris is a purple photosynthetic bacterium characterized by a versatile nature and a remarkable ability to adapt to various environments. In this work, we focused our attention to its membrane characteristics and defined the structural and conformational features of the O-chain polysaccharide of LPS isolated from R. palustris strain BisA53. This strain produces a polymer with a trisaccharide repeating unit characterized by d-rhamnose, 3-deoxy-d-lyxo-2-heptulosaric acid (Dha), and a novel C-branched monosaccharide, a 4-amino-4,6-dideoxy-3-C-methyl-2-O-methyl-α-l-glucopyranose whose absolute configuration has been determined by a combination of 2D NMR spectroscopy and molecular mechanic and dynamic simulation.
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http://dx.doi.org/10.1016/j.carbpol.2014.08.037DOI Listing
December 2014