Publications by authors named "Antonia De Felice"

6 Publications

  • Page 1 of 1

Development of a Specific and Sensitive HPAEC-PAD Method for Quantification of Vi Polysaccharide Applicable to other Polysaccharides Containing Amino Uronic Acids.

Anal Chem 2020 05 24;92(9):6304-6311. Epub 2020 Apr 24.

GSK Vaccines Institute for Global Health, Via Fiorentina 1, 53100 Siena, Italy.

Typhoid fever is a major cause of morbidity and mortality in developing countries. Vaccines based on the Vi capsular polysaccharide are licensed or in development against typhoid fever. Vi content is a critical quality attribute for vaccines release, to monitor their stability and to ensure appropriate immune response. Vi polysaccharide is a homopolymer of α-1,4--acetylgalactosaminouronic acid, O-acetylated at the C-3 position, resistant to the commonly used acid hydrolysis for sugar chain depolymerization before monomer quantification. We previously developed a quantification method based on strong alkaline hydrolysis followed by High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection analysis, but with low sensitivity and use for quantification of an unknown product coming from polysaccharide depolymerization. Here we describe the development of a method for Vi polysaccharide quantification based on acid hydrolysis with concomitant use of trifluoroacetic and hydrochloric acids. A Design of Experiment approach was used for the identification of the optimal hydrolysis conditions. The method is 100-fold more sensitive than the previous one, and specifically, resulting in the formation of a known product, confirmed to be the Vi monomer both de-O- and de-N-acetylated by mono- and bidimensional Nuclear Magnetic Resonance spectroscopy and mass spectrometry. Accuracy and precision were determined, and chromatographic conditions were improved to result in reduced time of analysis. This method will facilitate characterization of Vi-based vaccines. Furthermore, a similar approach has the potential to be extended to other polysaccharides containing 2-amino uronic acids, as already verified here for O-antigen, serotype 12F, and types 5 and 8 capsular polysaccharides.
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http://dx.doi.org/10.1021/acs.analchem.9b05107DOI Listing
May 2020

Structural investigation of the lipopolysaccharide O-chain isolated from Burkholderia fungorum strain DSM 17061.

Carbohydr Res 2016 Oct 9;433:31-5. Epub 2016 Jul 9.

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

Gram-negative bacteria exhibit lipopolysaccharides (LPSs) on their outer membrane surface. LPS is considered one of the most potent bacterial virulence factors. Here we report the elucidation of the LPS O-chain structure isolated from Burkholderia fungorum, a bacterium isolated from the white-rot fungus Phanerochaete chrysosporium that can act as a pathogen for plants and domesticated animals. The structure was determined by the employment of detailed chemical and NMR spectroscopy analyses as the following.
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http://dx.doi.org/10.1016/j.carres.2016.07.008DOI Listing
October 2016

Serotype O:8 isolates in the Yersinia pseudotuberculosis complex have different O-antigen gene clusters and produce various forms of rough LPS.

Innate Immun 2016 Apr 11;22(3):205-17. Epub 2016 Feb 11.

Department of Agriculture Sciences, University of Napoli, Portici, Italy

In Yersinia pseudotuberculosis complex, the O-antigen of LPS is used for the serological characterization of strains, and 21 serotypes have been identified to date. The O-antigen biosynthesis gene cluster and corresponding O-antigen structure have been described for 18, leaving O:8, O:13 and O:14 unresolved. In this study, two O:8 isolates were examined. The O-antigen gene cluster sequence of strain 151 was near identical to serotype O:4a, though a frame-shift mutation was found in ddhD, while No. 6 was different to 151 and carried the O:1b gene cluster. Structural analysis revealed that No. 6 produced a deeply truncated LPS, suggesting a mutation within the waaF gene. Both ddhD and waaF were cloned and expressed in 151 and No. 6 strains, respectively, and it appeared that expression of ddhD gene in strain 151 restored the O-antigen on LPS, while waaF in No. 6 resulted in an LPS truncated less severely but still without the O-antigen, suggesting that other mutations occurred in this strain. Thus, both O:8 isolates were found to be spontaneous O-antigen-negative mutants derived from other validated serotypes, and we propose to remove this serotype from the O-serotyping scheme, as the O:8 serological specificity is not based on the O-antigen.
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http://dx.doi.org/10.1177/1753425916631403DOI Listing
April 2016

The LPS O-Antigen in Photosynthetic Bradyrhizobium Strains Is Dispensable for the Establishment of a Successful Symbiosis with Aeschynomene Legumes.

PLoS One 2016 5;11(2):e0148884. Epub 2016 Feb 5.

IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR IRD/SupAgro/INRA/UM2/CIRAD, Campus International de Baillarguet, TA A-82/J, 34398 Montpellier Cedex 5, France.

The photosynthetic bradyrhizobia are able to use a Nod-factor independent process to induce nitrogen-fixing nodules on some semi-aquatic Aeschynomene species. These bacteria display a unique LPS O-antigen composed of a new sugar, the bradyrhizose that is regarded as a key symbiotic factor due to its non-immunogenic character. In this study, to check this hypothesis, we isolated mutants affected in the O-antigen synthesis by screening a transposon mutant library of the ORS285 strain for clones altered in colony morphology. Over the 10,000 mutants screened, five were selected and found to be mutated in two genes, rfaL, encoding for a putative O-antigen ligase and gdh encoding for a putative dTDP-glucose 4,6-dehydratase. Biochemical analysis confirmed that the LPS of these mutants completely lack the O-antigen region. However, no effect of the mutations could be detected on the symbiotic properties of the mutants indicating that the O-antigen region of photosynthetic Bradyrhizobium strains is not required for the establishment of symbiosis with Aeschynomene.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148884PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743980PMC
July 2016

Structure, genetics and function of an exopolysaccharide produced by a bacterium living within fungal hyphae.

Chembiochem 2015 Feb 21;16(3):387-92. Epub 2014 Dec 21.

Leibniz Institute for Natural Product Research and Infection Biology (HKI), Department of Biomolecular Chemistry, Beutenbergstrasse 11a, 07745 Jena (Germany).

The rice seedling blight fungus Rhizopus microsporus has an unusual symbiosis with a bacterium, Burkholderia rhizoxinica, which lives within the fungal cytosol and produces a potent phytotoxin that causes severe losses in agriculture. To gain insight into symbiosis factors we investigated the endosymbiont's exopolysaccharide (EPS), a secreted matrix that plays pivotal roles in mediating cell-environment interactions. By a combination of homo- and heteronuclear 2D NMR experiments, we elucidated a previously unknown EPS structure: a repeating tetrasaccharide unit bearing a nonstoichiometric acetyl group on a mannose residue. We also analyzed the EPS biosynthesis gene cluster and generated a targeted mutant to compare the phenotypes. Scanning electron microscope images revealed a reduced ability of the mutant to form extracellular polymers around cell aggregates. Phylogenetic analyses suggest that the symbiont's EPS genes are retained through evolutionary processes.
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http://dx.doi.org/10.1002/cbic.201402488DOI Listing
February 2015

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