Casillo, Angela (2016) Characterization and biological activity of bacterial glycoconjugates in cold adaptation. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Lingua: English
Title: Characterization and biological activity of bacterial glycoconjugates in cold adaptation.
Date: 29 March 2016
Number of Pages: 157
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Scuola di dottorato: Scienze chimiche
Dottorato: Scienze chimiche
Ciclo di dottorato: 28
Coordinatore del Corso di dottorato:
Corsaro, Maria MichelaUNSPECIFIED
Date: 29 March 2016
Number of Pages: 157
Uncontrolled Keywords: exopolysaccharide; cold-adaptation; lipopolysaccharide; Colwellia;
Settori scientifico-disciplinari del MIUR: Area 03 - Scienze chimiche > CHIM/06 - Chimica organica
Date Deposited: 11 Apr 2016 16:55
Last Modified: 02 Nov 2016 11:05


The cryosphere, covering about one-fifth of the surface of the Earth, comprises several components: snow, river and lake ice, sea ice, ice sheets, ice shelves, glaciers and ice caps, and frozen ground which exist, both on land and beneath the oceans (Vaughan DG, et al. 2013). All these habitats, combining the low temperature and the low liquid water activity, are challenging for all the forms of life (Casanueva et al., 2010). These extreme environments are inhabited by microorganisms of all three domains of life; in particular, cold-adapted microorganisms belong to Archea and Bacteria domains. To survive in these harsh life conditions, these microorganisms have developed many adaptation strategies, including the over-expression of cold-shock and heat-shock proteins, the presence of unsaturated and branched fatty acids that maintain membrane fluidity (Chattopadhyay et al., 2006), the different phosphorylation of membrane proteins and lipopolysaccharides (Ummarino et al., 2003; Corsaro et al., 2004; Carillo et al., 2013; Casillo et al., 2015), and the production of cold-active enzymes (Huston et al., 2004), antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), and cryoprotectants (Deming et al., 2009). Among cryoprotectants, carbohydrate-based extracellular polymeric substances (EPS) have a pivotal role in cold adaptation, as they form an organic network within the ice, modifying the structure of brine channels and contributing in the enrichment and retention of microrganisms in ice (Krembs et al., 2002; Krembs et al., 2011; Ewert et al., 2013). Macromolecules belonging to the external layer are fundamental in adaptation mechanisms, as for example the lipopolysaccharides (LPSs), which constitute the 75% of the outer membrane. LPSs have a structural role since increase the strength of bacterial cell envelope and mediate the contacts with the external environment. The general structure of an LPS is characterized by three distinct portions: the lipid A, composed of the typical glucosamine disaccharide backbone with different pattern of acylation on the two sugar residues, the core oligosaccharide, distinguishable in a inner core and a outer core, and the O-chain polysaccharide built up of oligosaccharide repeating units. This latter moiety can be absent, and in that case LPSs are named lipooligosaccharides (LOSs). Schematic representation of a lipopolysaccharide. Since the outer membrane of Gram-negative bacteria is constituted mainly by LPSs, it is reasonable to assume that structural changes could be present in these macromolecules isolated from cold-adapted bacteria. This work has been focused especially on three different psychrophilic microorganisms, that are considered models for the study of adaptive strategies to subzero lifestyle:  Colwellia psychrerythraea strain 34H  Psychrobacter arcticus 273-4  Pseudoalteromonas haloplanktis TAC125 In particular, LPS molecules from C. psychrerythraea 34H grown in different conditions, and from P.arcticus, have been purified and analyzed by NMR spectroscopy and mass spectrometry. By comparing the structures obtained, especially for core oligosaccharides, it is possible to speculate that all of them are characterized by high negative charge density. This negative charge is furnished either by phosphate groups, usually linked to Kdo and lipid A saccharidic residues, or by uronic acids. These characteristics have been already found in other LPSs from psychrophilic microorganisms (Corsaro et al., 2004; Corsaro et al., 2008; Carillo et al., 2011), suggesting that such structural elements contribute to the tightness of the outer-membrane and to the association of LPS molecules through divalent cations (Ca2+ and Mg2+). LOS structure from C.psychrerythraea 34H. LOS from P.arcticus 273-4. Starting from the core region of LOS from C. psychrerythraea, previously characterized (Carillo et al., 2013), the structure of lipid A was totally elucidated. The high heterogeneity of this structure, showed by the fatty acids analysis, was confirmed by the complexity of MS and MS/MS spectra. These experiments, indicated a variable state of acylation ranging from tetra- to hepta-acylated glycoforms. The lipid A moiety displayed a structure that is quite new among the LPSs. In fact, it shows the presence of unsaturated 3-hydroxy fatty acids, a feature that up to now is reported only for Agrobacterium tumefaciens (Silipo et al., 2004) and Vibrio fischeri (Philips et al., 2011). In particular, the structure of lipid A from Colwellia psychrerythraea 34H is very similar to that of Vibrio fischeri; in both structures, very intriguing is the presence of an unusual set of modifications at the secondary acylation site of the position 3 of GlcNI consisting of phosphoglycerol (GroP) differently substituted. The structural characterization of different exopolysaccharides produced by Colwellia psychrerythraea have also been reported. The capsular polysaccharide structure from C. psychrerythraea is composed of a tetrasaccharidic repeating unit containing two amino sugars and two uronic acids. The unique characteristic of the capsular polysaccharide is the presence of the α-aminoacid, threonine as substituent (Carillo et al., 2015). The decoration of the polysaccharide with threonines is particularly intriguing to consider. In fact, amino acid motifs are common and crucial for the interaction with ice in several different kinds of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) (Graether et al., 2000). Then, the molecular mechanic and dynamic calculations were performed, in collaboration with Prof. Randazzo of Department of Pharmacy; the computed model shows that the CPS seems to assume in the space a "zig-zag" flexible arrangement and that the overall structure can be imagined like a spatial repetition of an hairpin-like substructure, where the threonines are placed externally and available to interact with the ice. These results, the resemblance of our CPS structure to that of AFGPs, and the lack of sequence coding for a known AFP in the genome of C. psychrerythraea 34H prompted us to assay the purified polymer for ice recrystallization inhibition activity. This analysis, performed by Dr. Bayer-Giraldi, suggest that CPS interacts with ice and that it has an effect on recrystallization (Carillo et al., 2015). Colwellia psychrerythraea is also involved in the production of other two different exopolysaccharides (EPSs) with cryoprotectant activity: an acidic polysaccharide, named EPS, and a mannan. The EPS structure consists of a trisaccharidic repeating unit containing two galacturonic acids and one residue of 2-acetamido-2,6-dideoxy-D-glucose (Qui2NAc). Again, this structure shows the presence of an α-aminoacid, but in this case the decoration is represented by an alanine linked to the galacturonic acid residue. The chemical nature of the EPS is similar to that of the CPS, as it shows both galacto- and gluco-configured monosaccharides and aminoacids. Ice recrystallization inhibition activity, performed by Prof. Matthew Gibson, has been tested also for the EPS; the results show that also EPS has an effect on recrystallization, even if less marked with respect to the CPS. MD simulation was performed on a simplified model made up by five repeats of the trisaccharide basic unit and clearly revealed that the three central repeats adopt a fairly linear conformation that roughly resembles a left-handed helix. This conformation seems to be stabilized by a series of inter residue H-bond interactions. Of particular interest are three structural features: i) the amide nitrogen of quinovosamine, ii) the specific β-glycosidic linkage of this residue, and ii) the presence of the alanine attached to the galacturonic acid. The last exopolysaccharide produced by C. psychrerythraea at 4°C is a mannan, built up of a backbone of mannose α-(1→6) branched at C-2 with oligosaccharidic side chains, a common arrangement of mannans isolated from fungi and yeasts. The peculiarity of these structures is that some of these arms end with a β-glucose residue, and that some arms are cross-linked through phosphodiester bridges. The ice recrystallization inhibition assay showed that it is as active as the CPS produced by the same bacterium. Furthermore, C. psychrerythraea has been grown at two temperatures other than 4°C, in order to understand the variations, if any, in the structures of the saccharidic constituents. In particular, -2 and 8°C have been chosen. The cells extraction confirmed the presence of a rough-LPS (LOS) and sugar analysis suggested that the saccharidic composition is identical to that of the LOS from Colwellia grown at 4°C; this result was also confirmed by the MALDI spectrum of the partially deacylated LOS (LOS-OH). In contrast, GC-MS analysis showed a different composition, with a different composition of 3-hydroxy fatty acids, thus suggesting some differences in the lipid A structure. Furthermore, the presence of the CPS and EPS is confirmed when Colwellia is grown at 8 and -2°C, even if at these temperatures, the production of an additional polysaccharide (named CPS2) has been observed. The last one consists of a trisaccharidic repeating unit and unlike the others, it is not decorated with amino-acids. Interestingly, this polysaccharide displays very low ice recrystallization inhibition. Numerous prokaryotes, including Colwellia psychrerythraea 34 H, are able to accumulate large amounts of lipophilic compounds as inclusion bodies in the cytoplasm. Members of most genera synthesize polymeric lipids such as poly(3-hydroxybutyrate) (PHB) or other polyhydroxyalkanoates (PHAs). These compounds represent an attractive “green” alternatives to conventional petroleum-based plastics, finding application in various fields. The production of PHAs from C. psychrerythraea 34H has been tested in different growth conditions; up to now, the best condition was obtained at 4°C and 72h after inoculation, with the accumulation of 10% PHB and 90% of 3-hydroxyhexanoate. As Colwellia psychreeythraea, Psychrobacter arcticus 273-4 is involved in the production of a mannan polysaccharide The ice recrystallization inhibition activity has been tested for this polymer, and compared to that of mannans produced by C. psychreeythraea and by the yeast S. cerevisiae, a commercial product. The activity for Psychrobacter is higher than C. psychreeythraea, while the mannan produced by the yeast was found to be completely inactive. Intriguingly, the yeast polymer differs from that of P. arcticus and C. psychreeythraea for the lack of t-Glc residue and phosphates, thus suggesting that these structural features may be connected with the lack of activity. Psychrobacter arcticus is known in literature for the production of a capsule, when grown in presence of high salt concentration, that could be an adaptation mechanism (Ayala-del-Rio et al., 2010). The preliminary purification, revealed that the capsule polysaccharide is produced in very low amount, thus suggesting that the growth conditions are not so appropriate. Then, it will be necessary to maximize its production in order to fully characterize the CPS. The last cold-adapted microorganism, is P. haloplanktis TAC125, the cell-free supernatant of which presents an anti-biofilm activity against S. epidermidis (Papa et al., 2013). A purification procedure was set up and the analysis of an enriched fraction demonstrated that the anti-biofilm activity is due to a small molecule, that likely works as signal. The molecule, identified and compared with the corresponding standard, is new among those already reported in the literature.

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