Characterization and biological activity of bacterial glycoconjugates in cold adaptation.

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

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

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

A marine isolate of bacillus pumilus secretes a pumilacidin active against staphylococcus aureus

Producing antimicrobials is a common adaptive behavior shared by many microorganisms, including marine bacteria. We report that SF214, a marine-isolated strain of Bacillus pumilus, produces at least two different molecules with antibacterial activity: a molecule smaller than 3 kDa active against Staphylococcus aureus and a molecule larger than 10 kDa active against Listeria monocytogenes. We focused our attention on the anti-Staphylococcus molecule and found that it was active at a wide range of pH conditions and that its secretion was dependent on the growth phase, medium, and temperature. A mass spectrometry analysis of the size-fractionated supernatant of SF214 identified the small anti-Staphylococcus molecule as a pumilacidin, a nonribosomally synthesized biosurfactant composed of a mixture of cyclic heptapeptides linked to fatty acids of variable length. The analysis of the SF214 genome revealed the presence of a gene cluster similar to the srfA-sfp locus encoding the multimodular, nonribosomal peptide synthases found in other surfactant-producing bacilli. However, the srfA-sfp cluster of SF214 differed from that present in other surfactant-producing strains of B. pumilus by the presence of an insertion element previously found only in strains of B. safensis

Complete Characterization of the O-Antigen from the LPS of Aeromonas bivalvium

Aeromonas species are found in the aquatic environment, drinking water, bottled mineral water, and different types of foods, such as meat, fish, seafood, or vegetables. Some of these species are primary or opportunistic pathogens for invertebrates and vertebrates, including humans. Among the pathogenic factors associated with these species, there are the lipopolysaccharides (LPSs). LPSs are the major components of the external leaflet of Gram-negative bacterial outer membrane. LPS is a glycoconjugate, generally composed of three portions: lipid A, core oligosaccharide, and O-specific polysaccharide or O-antigen. The latter, which may be present (smooth LPS) or not (rough LPS), is the most exposed part of the LPS and is involved in the pathogenicity by protecting infecting bacteria from serum complement killing and phagocytosis. The O-antigen is a polymer of repeating oligosaccharide units with high structural variability, particularly the terminal sugar, that confers the immunological specificity to the O-antigen. In this study, we established the structure of the O-chain repeating unit of the LPS from Aeromonas bivalvium strain 868 ET (=CECT 7113T = LMG 23376T), a mesophilic bacterium isolated from cockles (Cardium sp.) and obtained from a retail market in Barcelona (Spain), whose biosynthesis core LPS cluster does not contain the waaE gene as most of Aeromonas species. After mild acid hydrolysis, the lipid A was removed by centrifugation and the obtained polysaccharide was fully characterized by chemical analysis and NMR spectroscopy. The polymer consists of a heptasaccharide repeating unit containing D-GalNAc, L-Rha, D-GlcNAc, and D-FucNAc residues

Reproducibility of the assessment of the Fränkel manoeuvre for the evaluation of sagittal skeletal discrepancies in Class II individuals

The Fränkel manoeuvre is a procedure by which the mandible of Class II individuals is postured forward in dental Class I relationship. The evaluation of the resulting facial profile provides information concerning the components determining the sagittal discrepancy. Data concerning the reproducibility of its assessment are not available. This study aimed to evaluate the intra-observer and inter-observer reproducibility of the assessment of the manoeuvre and to assess whether the amount of clinical experience affects its reproducibility

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 (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

Pentadecanal and pentadecanoic acid coatings reduce biofilm formation of Staphylococcus epidermidis on PDMS

Staphylococcus epidermidis is well known to be one of the major causes of infections related to medical devices, mostly due to its strong capacity to form device-associated biofilms. Nowadays, these infections represent a severe burden to the public health system and the necessity of novel antibacterial strategies for the treatment of these difficult-to-eradicate infections is urgent. The Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 was found to be able to produce an anti-biofilm molecule, the pentadecanal, active against S. epidermidis. In this work, we modified one of the most widely used silicone-based polymers, polydimethylsiloxane (PDMS), by adsorption of pentadecanal and its most promising derivative, pentadecanoic acid, on the PDMS surface. The biofilm formation of S. epidermidis RP62A on both untreated and modified PDMS was performed in a parallel plate flow chamber system, demonstrating the capability of the proposed anti-biofilm coatings to strongly reduce the biofilm formation. Furthermore, drug-release capacity and long-term efficacy (21 days) were also proven for the pentadecanoic acid coating

Structural characterization of core Region in Erwinia amylovora lipopolysaccharide.

Erwinia amylovora (E. amylovora) is the first bacterial plant pathogen described and demonstrated to cause fire blight, a devastating plant disease affecting a wide range of species including a wide variety of Rosaceae. In this study, we reported the lipopolysaccharide (LPS) core structure from E. amylovora strain CFBP1430, the first one for an E. amylovora highly pathogenic strain. The chemical characterization was performed on the mutants waaL (lacking only the O-antigen LPS with a complete LPS-core), wabH and wabG (outer-LPS core mutants). The LPSs were isolated from dry cells and analyzed by means of chemical and spectroscopic methods. In particular, they were subjected to a mild acid hydrolysis and/or a hydrazinolysis and investigated in detail by one and two dimensional Nuclear Magnetic Resonance (NMR) spectroscopy and ElectroSpray Ionization Fourier Transform-Ion Cyclotron Resonance (ESI FT-ICR) mass spectrometry

Anti-Biofilm Activity of a Long-Chain Fatty Aldehyde from Antarctic Pseudoalteromonas haloplanktis TAC125 against Staphylococcus epidermidis Biofilm

Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti-S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis

A multi-analytical approach to better assess the keratan sulfate contamination in animal origin chondroitin sulfate

Abstract Chondroitin sulfate is a glycosaminoglycan widely used as active principle of anti-osteoarthritis drugs and nutraceuticals, manufactured by extraction from animal cartilaginous tissues. During the manufacturing procedures, another glycosaminoglycan, the keratan sulfate, might be contemporarily withdrawn, thus eventually constituting a contaminant difficult to be determined because of its structural similarity. Considering the strict regulatory rules on the pureness of pharmaceutical grade chondrotin sulfate there is an urgent need and interest to determine the residual keratan sulfate with specific, sensitive and reliable methods. To pursue this aim, in this paper, for the first time, we set up a multi-analytical and preparative approach based on: i) a newly developed method by high performance anion-exchange chromatography with pulsed amperometric detection, ii) gas chromatography-mass spectrometry analyses, iii) size exclusion chromatography analyses coupled with triple detector array module and on iv) strong anion exchange chromatography separation. Varied KS percentages, in the range from 0.1 to 19.0% (w/w), were determined in seven pharmacopeia and commercial standards and nine commercial samples of different animal origin and manufacturers. Strong anion exchange chromatography profiles of the samples showed three or four different peaks. These peaks analyzed by high performance anion-exchange with pulsed amperometric detection and size exclusion chromatography with triple detector array, ion chromatography and by mono- or two-dimensional nuclear magnetic resonance revealed a heterogeneous composition of both glycosaminoglycans in terms of sulfation grade and molecular weight. High molecular weight species (>100 KDa) were also present in the samples that counted for chains still partially linked to a proteoglycan core