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University of Naples “Federico Ii” UNIVERSITY OF NAPLES “FEDERICO II” Faculty of Mathematical, Physical and Natural Sciences phD Thesis in Chemical Sciences XXV cycle Extremophile bacteria glycolipids: structure and biological activity SARA CARILLO Tutor Ch.ma Prof.ssa Maria Michela Corsaro Supervisor Ch.mo Prof. Giovanni Palumbo Alla mia famiglia… quella vecchia e quella nuova Summary Introduction Chapter One Life in extreme environments 3 1.1 Psychrophiles 5 1.2 Haloalkaliphiles 7 1.3 Industrial and biotechnological applications 9 Chapter Two Gram-negative Bacteria 13 2.1 The organization of the outer membrane 14 2.2 The lipopolysaccharides chemical features and structures 16 2.3 Exopolysaccharides: capsule and slime 19 2.4 The lipopolysaccharides: biological activity 21 Chapter Three Methodology in the Study of Lipopolysaccharides 27 3.1 Isolation and purification of LPSs 27 3.2 Chemical analysis and reactions on LPSs 29 3.2.1 O-chain structure determination 33 3.2.2 Lipid A structure determination 33 ii Extremophile bacteria glycolipids: structure and biological activity 3.2.3 Core region structure determination 34 3.3 Chromatography in the study of LPSs 35 3.4 Mass spectrometry in the study of LPSs 37 3.5 NMR spectroscopy in the study of LPSs 38 Psychrophiles Chapter Four Pseudoalteromonas haloplanktis TAB 23 47 4.1 Isolation and compositional analysis of the LOS 47 4.2 Isolation and characterization of the acid hydrolysis products 49 4.3 de-O-acylation of the LOS 51 4.4 de-N- acylation of the LOS-OH 52 4.5 Biological activities of lipid A 58 4.6 Conclusions 61 Chapter Five Colwellia psychrerythraea 34H 65 5.1 LPS extraction and preliminary analysis 66 5.2 Mass spectrometric analysis of the O-deacylated LOSPCP 67 5.3 NMR analysis of the fully deacylated LOSPCP 69 Summary iii 5.4 NMR analysis of the LOS-OH 74 5.5 Lipid A structure elucidation and biological assay 77 5.6 The capsular polysaccharide of C. psychrerythraea 79 5.7 Conclusions 84 Psychrophiles: Conclusions 88 Haloalkaliphiles Chapter Six Halomonas alkaliantarctica strain CRSS 95 6.1 Isolation and compositional analysis of LOS fraction 96 6.2 Analysis of the deacylated products 97 6.3 Conclusions 106 Chapter Seven Halomonas stevensii strain S18214 109 7.1 LPS extraction and purification 110 7.2 O-chain polysaccharide structural determination 111 7.3 Mass spectrometric analysis of de-O-acylated LPS 114 7.4 NMR characterization of OS1 117 7.5 Lipid A structural characterization 123 7.6 Conclusions 128 iv Extremophile bacteria glycolipids: structure and biological activity Chapter Eight Salinivibrio sharmensis strain BAGT 133 8.1 LPS extraction and purification 133 8.2 Mass spectrometric analysis of the deacylated LPS 134 8.3 NMR characterization of OSE and OSC 137 8.4 Lipid A characterization 143 8.5 Conclusions 148 Haloalkaliphiles: Conclusions 152 Experimental Part Chapter Nine Materials and Methods 157 9.1 Bacteria growth 157 9.1.1 Halomonas alkaliantarctica strain CRSS 157 9.1.2 Halomonas stevensii strain S18214 157 9.1.3 Salinivibrio sharmensis strain BAGT 158 9.1.4 Pseudoalteromonas haloplanktis strain TAB 23 158 9.1.5 Colwellia psychrerythraea strain 34H 158 9.2 General and analytical method 159 9.2.1 LPS extraction and purification 159 9.2.2 Electrophoretic analysis 160 Summary v 9.2.3 Chemical analysis 160 9.2.4 Mild acid hydrolysis 162 9.2.5 de-O and de-N-acylation of LPSs 162 9.2.6 Ammonium hydroxide hydrolysis of lipid A 163 9.2.7 CPS isolation and purification 163 9.3 HPAEC-PAD 163 9.4 Mass spectrometry 164 9.5 NMR spectroscopy 165 9.6 Biological assays 166 Bibliography 169 vi Extremophile bacteria glycolipids: structure and biological activity Abbreviations AA Alditol Acetate AFPs Antifreeze Proteins Ala L-Alanine AMG acetylated O-methyl glycosides CD14 Cluster of differentiation 14 Col 3,6-dideoxy-L-xylo-hexose CPS Capsular Polysaccharide CSD Capillary Skimmed Dissociation D,D-Hep D-glycero-D-manno-heptose DOC-PAGE DeOxyCholate PolyAcrylamide Gel Electrophoresis DQF-COSY Double Quantum Filter-Correlation SpectroscopY EPS Exopolysaccharide ElectroSpray Ionization Fourier Transform-Ion ESI FT-ICR Cyclotron Resonance Et3N Triethilamine GAG GlycosAmino Glycans Gal D-galactose GalA D-galacturonic acid GalN 2-deoxy-2-amino-D-galctose GC-MS Gas Chromatography interfaced with Mass Spectrometry Glc D-glucose GlcA D-glucuronic acid GlcN 2-deoxy-2-amino-D-glucose Gro Glycerol HMBC Heteronuclear Multiple Bond Correlation High Pressure Anion Exchange Chromatography – HPAEC-PAD Pulsed Amperometric Detection HPLC High Performance Liquid Chromatography Summary vii HSQC Heteronuclear Single Quantum Correlation IL-6 Interleukin-6 IRMPD InfraRed MultiPhoton Dissociation Kdo 3-deoxy-D-manno-oct-2-ulosonic acid L,D-Hep L-glycero-D-manno-heptose LOS Lipooligosaccharide LPS Lipopolysaccharide Mal Myelin and lymphocyte protein Matrix Assisted Laser Desorption Ionization – Time Of MALDI-TOF Flight Man D-mannose MD-2 myeloid differentiation factor 2 MPLA monophosphoryl lipid A MyD88 Myeloid differentiation primary response gene (88) NMR Nuclear Magnetic Resonance NOE Nuclear Overhauser Effect -3 P -PO4 PAMPs Pathogen Associated Molecular Patterns PCP Petroleum Ether/Chloroform/Phenol 90% PMAA Partially Methylated Alditol Acetate Rha 6-deoxy-L-mannose ROESY Rotating frame Overhauser Effect Spectroscopy Sodium Dodecyl Sulphate PolyAcrylamide Gel SDS-PAGE Electrophoresis TEM Trasmission Electronic Microscopy TFA trifluoroacetic acid THP-1 Human acute monocytic leukemia cell line Thr L-Threonine TIR Toll-IL-1 receptor TLRs Toll-like receptors TNFα Tumor necrosis factor α viii Extremophile bacteria glycolipids: structure and biological activity TOCSY Total Correlation Spectroscopy TRAM TRIF-related adaptor molecule TRIF TIR-domain-containing adapter-inducing interferon-β ZPS Zwitterionic polysaccharide Abstract _________________________________________________________________________________________ Extremophile bacteria are able to survive in harsh life conditions, such as high or low temperatures (thermophiles and psychrophiles, respectively), high pressure (barophiles), high or low pH values (acidophiles or alkalophiles), environments characterized by high salt concentrations (halophiles). Structural features of the macromolecules belonging to the external layer are fundamental in adaptation mechanisms, e.g. it is well known that halophiles membrane phospholipids showed an increased negative charge density, while in psychrophiles these molecules display shorter acyl chains and higher unsaturation degree. In Gram-negative bacteria, 75% of the outer membrane is constituted by lipopolysaccharides (LPSs). Consequently they play a key role in the adaptation and survival in extreme life conditions. Nevertheless, very few LPSs isolated from extremophilic bacteria has been characterized so far. LPS are constituted by three covalently linked regions: - lipid A, which is the glycolipidic portion of the macromolecule. It is the most conservative region between bacteria belonging to the same genus and represents the minimal endotoxic structural motif; - core region, which is an oligosaccharidic portion where it is possible to find LPSs peculiar monosaccharides, such as heptoses and Kdo (3-deoxy oct-2-oulosonic acid); - O-chain, which is the polysaccharidic region, not always expressed by the bacterium. Moreover, O-chain is highly variable even among bacteria belonging to the same species. Beside the structural characterization of LPS, aimed at adaptation mechanisms comprehension, also their biological activity is worth being investigated. 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