Publications by authors named "Immacolata Speciale"

21 Publications

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Structural specificities of cell surface β-glucan polysaccharides determine commensal yeast mediated immuno-modulatory activities.

Nat Commun 2021 06 14;12(1):3611. Epub 2021 Jun 14.

Division of Integrative Biosciences and Biotechnology, Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea.

Yeast is an integral part of mammalian microbiome, and like commensal bacteria, has the potential of being harnessed to influence immunity in clinical settings. However, functional specificities of yeast-derived immunoregulatory molecules remain elusive. Here we find that while under steady state, β-1,3-glucan-containing polysaccharides potentiate pro-inflammatory properties, a relatively less abundant class of cell surface polysaccharides, dubbed mannan/β-1,6-glucan-containing polysaccharides (MGCP), is capable of exerting potent anti-inflammatory effects to the immune system. MGCP, in contrast to previously identified microbial cell surface polysaccharides, through a Dectin1-Cox2 signaling axis in dendritic cells, facilitates regulatory T (Treg) cell induction from naïve T cells. Furthermore, through a TLR2-dependent mechanism, it restrains Th1 differentiation of effector T cells by suppressing IFN-γ expression. As a result, administration of MGCP display robust suppressive capacity towards experimental inflammatory disease models of colitis and experimental autoimmune encephalomyelitis (EAE) in mice, thereby highlighting its potential therapeutic utility against clinically relevant autoimmune diseases.
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http://dx.doi.org/10.1038/s41467-021-23929-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8203763PMC
June 2021

Aberrant sialylation in a patient with a variant and liver adenomatosis.

iScience 2021 Apr 18;24(4):102323. Epub 2021 Mar 18.

CNR, Institute for Polymers, Composites and Biomaterials, IPCB, 95126 Catania, Italy.

Glycosylation is a fundamental post-translational modification of proteins that boosts their structural diversity providing subtle and specialized biological properties and functions. All those genetic diseases due to a defective glycan biosynthesis and attachment to the nascent glycoproteins fall within the wide area of congenital disorders of glycosylation (CDG), mostly causing multisystem involvement. In the present paper, we detailed the unique serum N-glycosylation of a CDG-candidate patient with an unexplained neurological phenotype and liver adenomatosis harboring a recurrent pathogenic variant. Serum transferrin isoelectric focusing showed a surprising N-glycosylation pattern consisting on hyposialylation, as well as remarkable hypersialylation. Mass spectrometry-based glycomic analyses of individual serum glycoproteins enabled to unveil hypersialylated complex N-glycans comprising up to two sialic acids per antenna. Further advanced MS analysis showed the additional sialic acid is bonded through an α2-6 linkage to the peripheral N-acetylglucosamine residue.
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http://dx.doi.org/10.1016/j.isci.2021.102323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050382PMC
April 2021

Structural characterisation of the oligosaccharide from Moraxella bovoculi type strain 237 (ATCC BAA-1259) lipooligosaccharide.

Carbohydr Res 2021 May 19;503:108293. Epub 2021 Mar 19.

School of Medical Science, Griffith University, Gold Coast Campus, Queensland, 4222, Australia. Electronic address:

The Gram-negative bacterium Moraxella bovoculi is associated with infectious bovine keratoconjunctivitis (IBK), colloquially known as 'pink-eye'. IBK is an extremely contagious ocular disease of cattle. We report here the structure of the oligosaccharide derived from the lipooligosaccharide from M. bovoculi type strain 237 (also known as ATCC BAA-1259T). GLC-MS and correlation NMR analysis of the oligosaccharide revealed 5 sugar residues, with a notable central branched 3,4,6-α-D-Glcp. An additional α-D-Manp was present ~30% on the sub-terminal α-D-Manp of the 4-linked branch. This oligosaccharide structure was consistent with other members of the Moraxellaceae where no heptose was present and 5-linked Kdo was directly attached to the central 3,4,6-α-D-Glcp.
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http://dx.doi.org/10.1016/j.carres.2021.108293DOI Listing
May 2021

Lipopolysaccharide from Gut-Associated Lymphoid-Tissue-Resident Alcaligenes faecalis: Complete Structure Determination and Chemical Synthesis of Its Lipid A.

Angew Chem Int Ed Engl 2021 04 22;60(18):10023-10031. Epub 2021 Mar 22.

Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan.

Alcaligenes faecalis is the predominant Gram-negative bacterium inhabiting gut-associated lymphoid tissues, Peyer's patches. We previously reported that an A. faecalis lipopolysaccharide (LPS) acted as a weak agonist for Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) receptor as well as a potent inducer of IgA without excessive inflammation, thus suggesting that A. faecalis LPS might be used as a safe adjuvant. In this study, we characterized the structure of both the lipooligosaccharide (LOS) and LPS from A. faecalis. We synthesized three lipid A molecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1- and 4'-phosphates. Hexaacylated A. faecalis lipid A showed moderate agonistic activity towards TLR4-mediated signaling and the ability to elicit a discrete interleukin-6 release in human cell lines and mice. It was thus found to be the active principle of the LOS/LPS and a promising vaccine adjuvant candidate.
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http://dx.doi.org/10.1002/anie.202012374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8252424PMC
April 2021

Chlorovirus PBCV-1 Multidomain Protein A111/114R Has Three Glycosyltransferase Functions Involved in the Synthesis of Atypical N-Glycans.

Viruses 2021 Jan 10;13(1). Epub 2021 Jan 10.

Nebraska Center for Virology, University of Nebraska, Lincoln, NE 68583-0900, USA.

The structures of the four -linked glycans from the prototype chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of genetic and structural analyses indicates that the protein coded by PBCV-1 gene , conserved in all chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the chloroviruses do not use the canonical host endoplasmic reticulum-Golgi glycosylation pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of cellular organelles using virus-encoded enzymes.
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http://dx.doi.org/10.3390/v13010087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826918PMC
January 2021

Carbohydrate-based adjuvants.

Drug Discov Today Technol 2020 Dec 9;35-36:57-68. Epub 2020 Oct 9.

Department of Agricultural Sciences, University of Naples Federico II, Via Università, 100, 80055 Portici (NA), Italy. Electronic address:

Carbohydrate adjuvants are safe and biocompatible compounds usable as sustained delivery systems and stimulants of ongoing humoral and cellular immune responses, being especially suitable for the development of vaccines against intracellular pathogens where alum is useless. The development of new adjuvants is difficult and expensive, however, in the last two years, seven new carbohydrate-based adjuvants have been patented, also there are twelve ongoing clinical trials of vaccines that contain carbohydrate-based adjuvants, as well as numerous publications on their mechanism of action and safety. More research is necessary to improve the existent adjuvants and develop innovative ones.
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http://dx.doi.org/10.1016/j.ddtec.2020.09.005DOI Listing
December 2020

Structure of the O-Antigen and the Lipid A from the Lipopolysaccharide of Fusobacterium nucleatum ATCC 51191.

Chembiochem 2021 Apr 14;22(7):1252-1260. Epub 2020 Dec 14.

Department of Agricultural Sciences, University of Naples Federico II, Via Università, 100, 80055, Portici NA, Italy.

Fusobacterium nucleatum is a common member of the oral microbiota. However, this symbiont has been found to play an active role in disease development. As a Gram-negative bacterium, F. nucleatum has a protective outer membrane layer whose external leaflet is mainly composed of lipopolysaccharides (LPSs). LPSs play a crucial role in the interaction between bacteria and the host immune system. Here, we characterised the structure of the O-antigen and lipid A from F. nucleatum ssp. animalis ATCC 51191 by using a combination of GC-MS, MALDI and NMR techniques. The results revealed a novel repeat of the O-antigen structure of the LPS, [→4)-β-d-GlcpNAcA-(1→4)-β-d-GlcpNAc3NAlaA-(1→3)-α-d-FucpNAc4NR-(1→], (R=acetylated 60 %), and a bis-phosphorylated hexa-acylated lipid A moiety. Taken together these data showed that F. nucleatum ATCC 51191 has a distinct LPS which might differentially influence recognition by immune cells.
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http://dx.doi.org/10.1002/cbic.202000751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048906PMC
April 2021

Chlorovirus PBCV-1 protein A064R has three of the transferase activities necessary to synthesize its capsid protein N-linked glycans.

Proc Natl Acad Sci U S A 2020 11 2;117(46):28735-28742. Epub 2020 Nov 2.

Department of Agricultural Sciences, University of Napoli Federico II, 80055 Portici NA, Italy;

chlorella virus-1 (PBCV-1) is a large double-stranded DNA (dsDNA) virus that infects the unicellular green alga NC64A. Unlike many other viruses, PBCV-1 encodes most, if not all, of the enzymes involved in the synthesis of the glycans attached to its major capsid protein. Importantly, these glycans differ from those reported from the three domains of life in terms of structure and asparagine location in the sequon of the protein. Previous data collected from 20 PBCV-1 spontaneous mutants (or antigenic variants) suggested that the gene encodes a glycosyltransferase (GT) with three domains, each with a different function. Here, we demonstrate that: domain 1 is a β-l-rhamnosyltransferase; domain 2 is an α-l-rhamnosyltransferase resembling only bacterial proteins of unknown function, and domain 3 is a methyltransferase that methylates the C-2 hydroxyl group of the terminal α-l-rhamnose (Rha) unit. We also establish that methylation of the C-3 hydroxyl group of the terminal α-l-Rha is achieved by another virus-encoded protein A061L, which requires an O-2 methylated substrate. This study, thus, identifies two of the glycosyltransferase activities involved in the synthesis of the -glycan of the viral major capsid protein in PBCV-1 and establishes that a single protein A064R possesses the three activities needed to synthetize the 2-OMe-α-l-Rha-(1→2)-β-l-Rha fragment. Remarkably, this fragment can be attached to any xylose unit.
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http://dx.doi.org/10.1073/pnas.2016626117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682578PMC
November 2020

Selection of Exopolysaccharide-Producing () Isolated from Algerian Fermented Foods for the Manufacture of Skim-Milk Fermented Products.

Microorganisms 2020 Jul 23;8(8). Epub 2020 Jul 23.

Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias-Consejo Superior de Investigaciones Científicas (IPLA-CSIC), 33300 Villaviciosa, Asturias, Spain.

The exopolysaccharide (EPS)-producing (renamed as ) LBIO1, LBIO14 and LBIO28 strains, isolated from fermented dairy products typical from Algeria, were characterized to evaluate the impact of the polymers in milk fermentations. Their genomes revealed the presence of two complete clusters of the four described for the reference strain WCFS1. Besides, the three strains presented identical sequences of and clusters, but LBIO1 and LBIO28 harbour three genes belonging to which are absent in the LBIO14 genome. The EPS purified from fermented skim-milks manufactured with the strains showed identical nuclear magnetic resonance (H-NMR) and size exclusion chromatography coupled with a multiangle laser light scattering detector (SEC-MALLS) profiles for polymers LBIO1 and LBIO28, whereas LBIO14 EPS was different due to the lack of the high-molecular weight (HMW)-EPS and the absence of specific monosaccharide's peaks in the anomeric region of its proton NMR spectrum. The presence of the HMW-EPS correlated with optimal sensorial-physical characteristics of the fermented skim-milks (ropy phenotype). Their microstructures, studied by confocal scanning laser microscopy (CSLM), also showed differences in the organization of the casein-network and the distribution of the bacteria inside this matrix. Therefore, the strain LBIO1 can be proposed for the manufacture of dairy products that require high whey retention capability, whereas LBIO28 could be applied to increase the viscosity.
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http://dx.doi.org/10.3390/microorganisms8081101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465087PMC
July 2020

Structure of the unusual HH103 lipopolysaccharide and its role in symbiosis.

J Biol Chem 2020 08 16;295(32):10969-10987. Epub 2020 Jun 16.

Department of Microbiology, Faculty of Biology, University of Seville, Sevilla, Spain

Rhizobia are soil bacteria that form important symbiotic associations with legumes, and rhizobial surface polysaccharides, such as K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS), might be important for symbiosis. Previously, we obtained a mutant of HH103, , that does not produce KPS, a homopolysaccharide of a pseudaminic acid derivative, but whose LPS electrophoretic profile was indistinguishable from that of the WT strain. We also previously demonstrated that the HH103 operon is responsible for 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-l--l--nonulosonic acid [Pse5NAc7(3OHBu)] production and is involved in HH103 KPS and LPS biosynthesis and that an HH103 mutant cannot produce KPS and displays an altered LPS structure. Here, we analyzed the LPS structure of HH103 , focusing on the carbohydrate portion, and found that it contains a highly heterogeneous lipid A and a peculiar core oligosaccharide composed of an unusually high number of hexuronic acids containing β-configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its α-configuration, was the only structural component of the HH103 KPS and, to the best of our knowledge, has never been reported from any other rhizobial LPS. We also show that Pse5NAc7(3OHBu) is the complete or partial epitope for a mAb, NB6-228.22, that can recognize the HH103 LPS, but not those of most of the strains tested here. We also show that the LPS from HH103 is identical to that of HH103 but devoid of any Pse5NAc7(3OHBu) residues. Notably, this mutant was severely impaired in symbiosis with its host, .
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http://dx.doi.org/10.1074/jbc.RA120.013393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415993PMC
August 2020

Biopolymer Skeleton Produced by Rhizobium radiobacter: Stoichiometric Alternation of Glycosidic and Amidic Bonds in the Lipopolysaccharide O-Antigen.

Angew Chem Int Ed Engl 2020 04 10;59(16):6368-6374. Epub 2020 Mar 10.

Department of Agricultural Sciences, University of Napoli, Via Università 100, 80055, Portici (NA), Italy.

The lipopolysaccharide (LPS) O-antigen structure of the plant pathogen Rhizobium radiobacter strain TT9 and its possible role in a plant-microbe interaction was investigated. The analyses disclosed the presence of two O-antigens, named Poly1 and Poly2. The repetitive unit of Poly2 constitutes a 4-α-l-rhamnose linked to a 3-α-d-fucose residue. Surprisingly, Poly1 turned out to be a novel type of biopolymer in which the repeating unit is formed by a monosaccharide and an amino-acid derivative, so that the polymer has alternating glycosidic and amidic bonds joining the two units: 4-amino-4-deoxy-3-O-methyl-d-fucose and (2'R,3'R,4'S)-N-methyl-3',4'-dihydroxy-3'-methyl-5'-oxoproline). Differently from the O-antigens of LPSs from other pathogenic Gram-negative bacteria, these two O-antigens do not activate the oxidative burst, an early innate immune response in the model plant Arabidopsis thaliana, explaining at least in part the ability of this R. radiobacter strain to avoid host defenses during a plant infection process.
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http://dx.doi.org/10.1002/anie.201914053DOI Listing
April 2020

Structural features and immunological perception of the cell surface glycans of Lactobacillus plantarum: a novel rhamnose-rich polysaccharide and teichoic acids.

Carbohydr Polym 2020 Apr 10;233:115857. Epub 2020 Jan 10.

Task Force on Microbiome Studies, University of Naples Federico II, Naples, Italy; Department of Agricultural Sciences, University of Naples, 80055 Portici, Italy. Electronic address:

The capsular material from Lactobacillus plantarum IMB19, an isolate from fermented vegetables, has been analyzed and our results demonstrate that most of the coat of this bacterium consists of glycerol- and ribitol-type teichoic acids, further decorated with other substituents (α-glucose and alanine), and of a capsular polysaccharide (CPS) with a linear nonasaccharide repeating unit, rich in rhamnose, interconnected to the next via a phosphodiester bridge. Stimulation of immune cells with the total capsular material resulted in the enhancement of immunostimulatory (IFNγ, TNF-α, IL-6 and IL-12) or immuno-regulatory (IL-10) cytokines in an in vitro splenocyte culture system. The capsular polysaccharide, and not the teichoic acids mixture, was responsible for the IFNγ production. Indeed, a significant increase of IFNγ along with other inflammatory cytokines, and a dose response in IFNγ expression with an EC of 3.16 μM was found for CPS, disclosing that this polysaccharide is a potent immunostimulatory molecule.
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http://dx.doi.org/10.1016/j.carbpol.2020.115857DOI Listing
April 2020

Bifidobacterium bifidum presents on the cell surface a complex mixture of glucans and galactans with different immunological properties.

Carbohydr Polym 2019 Aug 3;218:269-278. Epub 2019 May 3.

Task Force on Microbiome Studies, University of Naples Federico II, Naples, Italy; Department of Agricultural Sciences, University of Naples, 80055, Portici, Italy. Electronic address:

The chemical structure of cell surface polysaccharides isolated from Bifidobacterium bifidum strain PRI1, an important member of the gut microbiota of breast-fed infants, has been elucidated by chemical and NMR spectroscopy analysis. Results demonstrated that the bacterium produces a complex mixture of polysaccharides that could be classified in two main groups: a phospho-glycero-β-galactofuranan, PGβG, and a mixture composed of four neutral polysaccharides named as (CSGG), composed of β-(1 → 6)-glucan, β-(1 → 4)-galactan, β-(1 → 6)-galactan, β-galactofuranan and starch. These two fractions exerted different immune responses when assayed on dendritic cells: PGβG enhanced pro-inflammatory immune responses by increasing interferon-γ levels while CSGG induced immunosuppressive regulatory T cells and interleukin-10. These findings demonstrate that bacterial polysaccharides have a distinct role depending on their chemical structure in regulation of the host/bacterium interaction. Our findings suggest that polysaccharides may differentially regulate the host immunity depending on the composition of this complex mixture, either enhancing immunity or inducing immune tolerance.
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http://dx.doi.org/10.1016/j.carbpol.2019.05.006DOI Listing
August 2019

The -glycan structures of the antigenic variants of chlorovirus PBCV-1 major capsid protein help to identify the virus-encoded glycosyltransferases.

J Biol Chem 2019 04 8;294(14):5688-5699. Epub 2019 Feb 8.

From the Department of Agricultural Sciences, University of Napoli Federico II, Via Università 100, 80055 Portici NA, Italy,

The chlorovirus chlorella virus 1 (PBCV-1) is a large dsDNA virus that infects the microalga NC64A. Unlike most other viruses, PBCV-1 encodes most, if not all, of the machinery required to glycosylate its major capsid protein (MCP). The structures of the four -linked glycans from the PBCV-1 MCP consist of nonasaccharides, and similar glycans are not found elsewhere in the three domains of life. Here, we identified the roles of three virus-encoded glycosyltransferases (GTs) that have four distinct GT activities in glycan synthesis. Two of the three GTs were previously annotated as GTs, but the third GT was identified in this study. We determined the GT functions by comparing the WT glycan structures from PBCV-1 with those from a set of PBCV-1 spontaneous GT gene mutants resulting in antigenic variants having truncated glycan structures. According to our working model, the virus gene encodes a GT with three domains: domain 1 has a β-l-rhamnosyltransferase activity, domain 2 has an α-l-rhamnosyltransferase activity, and domain 3 is a methyltransferase that decorates two positions in the terminal α-l-rhamnose (Rha) unit. The gene encodes a β-xylosyltransferase that attaches the distal d-xylose (Xyl) unit to the l-fucose (Fuc) that is part of the conserved -glycan core region. Last, gene encodes a GT that is involved in the attachment of a semiconserved element, α-d-Rha, to the same l-Fuc in the core region. Our results uncover GT activities that assemble four of the nine residues of the PBCV-1 MCP -glycans.
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http://dx.doi.org/10.1074/jbc.RA118.007182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462530PMC
April 2019

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

Acinetobacter baumannii K13 and K73 capsular polysaccharides differ only in K-unit side branches of novel non-2-ulosonic acids: di-N-acetylated forms of either acinetaminic acid or 8-epiacinetaminic acid.

Carbohydr Res 2017 Nov 19;452:149-155. Epub 2017 Oct 19.

N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia.

Structures of capsular polysaccharides of Acinetobacter baumannii isolates carrying KL13 and KL73 gene clusters were established. The closely related KL73 and KL13 gene clusters differ only by one gene in the module responsible for synthesis of the non-2-ulosonic acids. The K13 and K73 polysaccharides differ only in a single side-chain sugar, which is either 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-altro- or -d-glycero-l-altro-non-2-ulosonic acid [di-N-acetylated forms of acinetaminic acid (Aci5Ac7Ac) or 8-epiacinetaminic acid (8eAci5Ac7Ac), respectively]. The KL13 also is closely related to the KL12 gene cluster, which contains a different wzy gene encoding the K unit polymerase. Accordingly, the otherwise near identical K units are linked differently via an α-d-FucpNAc-(1 → 4)-d-Galp linkage in K13 and K73 or an α-d-FucpNAc-(1 → 3)-d-GalpNAc linkage in K12. This finding confirms the predicted substrate of the ItrB3 initiating transferase as d-FucpNAc. Glycosyltransferases predicted to catalyse the linkage of d-Galp or d-GalpNAc to l-FucpNAc in the growing K13 and K73 or K12 units, respectively, differ by only two amino acids.
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http://dx.doi.org/10.1016/j.carres.2017.10.005DOI Listing
November 2017

Structure of the N-glycans from the chlorovirus NE-JV-1.

Antonie Van Leeuwenhoek 2017 Nov 22;110(11):1391-1399. Epub 2017 Mar 22.

Department of Agricultural Sciences, University of Napoli, 80055, Portici, Italy.

Results from recent studies are breaking the paradigm that all viruses depend on their host machinery to glycosylate their proteins. Chloroviruses encode several genes involved in glycan biosynthesis and some of their capsid proteins are decorated with N-linked oligosaccharides with unique features. Here we describe the elucidation of the N-glycan structure of an unusual chlorovirus, NE-JV-1, that belongs to the Pbi group. The host for NE-JV-1 is the zoochlorella Micractinium conductrix. Spectroscopic analyses established that this N-glycan consists of a core region that is conserved in all of the chloroviruses. The one difference is that the residue 3OMe-L-rhamnose is acetylated at the O-2 position in a non-stoichiometric fashion.
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http://dx.doi.org/10.1007/s10482-017-0861-3DOI Listing
November 2017

Characterization of a new chlorovirus type with permissive and non-permissive features on phylogenetically related algal strains.

Virology 2017 01 27;500:103-113. Epub 2016 Oct 27.

Nebraska Center for Virology, University of Nebraska-Lincoln, NE 68583-0900, USA; Department of Plant Pathology and University of Nebraska-Lincoln, NE 68583-0722, USA. Electronic address:

A previous report indicated that prototype chlorovirus PBCV-1 replicated in two Chlorella variabilis algal strains, NC64A and Syngen 2-3, that are ex-endosymbionts isolated from the protozoan Paramecium bursaria. Surprisingly, plaque-forming viruses on Syngen 2-3 lawns were often higher than on NC64A lawns from indigenous water samples. These differences led to the discovery of viruses that exclusively replicate in Syngen 2-3 cells, named Only Syngen (OSy) viruses. OSy-NE5, the prototype virus for the proposed new species, had a linear dsDNA genome of 327kb with 44-nucleotide-long, incompletely base-paired, covalently closed hairpin ends. Each hairpin structure was followed by an identical 2612 base-paired inverted sequence after which the DNA sequence diverged. OSy-NE5 encoded 357 predicted CDSs and 13 tRNAs. Interestingly, OSy-NE5 attached to and initiated infection in NC64A cells but infectious progeny viruses were not produced; thus OSy-NE5 replication in NC64A is blocked at some later stage of replication.
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http://dx.doi.org/10.1016/j.virol.2016.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127778PMC
January 2017

Structure of repeating unit of the capsular polysaccharide from Acinetobacter baumannii D78 and assignment of the K4 gene cluster.

Carbohydr Res 2016 Nov 15;434:12-17. Epub 2016 Jul 15.

Department of Agricultural Sciences, University of Napoli, Italy. Electronic address:

The structure of the K4 capsular polysaccharide (CPS) from a multiply antibiotic resistant Acinetobacter baumannii isolate D78 was elucidated by chemical and spectroscopical analysis. The K4 repeating unit is composed entirely by aminosugars and the trisaccharide repeating unit of the backbone polysaccharide is branched with a terminal N-acetyl-galactosamine capped with pyruvate as cyclic acetal. Each residue is in the pyranose form, α configured at the anomeric center, and has the D absolute configuration. The genes responsible for the synthesis of the K4 repeat unit and its polymerization and export are found in the capsule locus KL4 present in D78. The pyruvate acetal addition to galactosamine is formed by Ptr1, a novel pyruvate transferase, encoded at this locus.
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http://dx.doi.org/10.1016/j.carres.2016.07.016DOI Listing
November 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

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
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