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1. (Article ID: 5732)
 
Bellich B, Jou IA, Caterino M, Rizzo R, Ravenscroft N, Fazli M, Tolker-Nielsen T, Brady JW, Cescutti P
Burkholderia cenocepacia H111 Produces a Water-Insoluble Exopolysaccharide in Biofilm: Structural Determination and Molecular Modelling
International Journal of Molecular Sciences 21(5) (2020) 1702
 

Biofilms are a multicellular way of life, where bacterial cells are close together and embedded in a hydrated macromolecular matrix which offers a number of advantages to the cells. Extracellular polysaccharides play an important role in matrix setup and maintenance. A water-insoluble polysaccharide was isolated and purified from the biofilm produced by Burkholderia cenocepacia strain H111, a cystic fibrosis pathogen. Its composition and glycosidic linkages were determined using Gas-Liquid Chromatography-Mass Spectrometry (GLC-MS) on appropriate carbohydrate derivatives while its complete structure was unraveled by 1D and 2D NMR spectroscopy in deuterated sodium hydroxide (NaOD) aqueous solutions. All the collected data demonstrated the following repeating unit for the water-insoluble B. cenocepacia biofilm polysaccharide: [3)-α-d-Galp-(1→3)-α-d-Glcp-(1→3)-α-d-Galp-(1→3)-α-d-Manp-(1→]n Molecular modelling was used, coupled with NMR Nuclear Overhauser Effect (NOE) data, to obtain information about local structural motifs which could give hints about the polysaccharide insolubility. Both modelling and NMR data pointed at restricted dynamics of local conformations which were ascribed to the presence of inter-residue hydrogen bonds and to steric restrictions. In addition, the good correlation between NOE data and calculated interatomic distances by molecular dynamics simulations validated potential energy functions used for calculations.

NMR, molecular modelling, polysaccharide structure, biofilm exopolysaccharides, Burkholderia cenocepacia H111

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2. (Article ID: 6203)
 
Bellich B, Teran LC, Fazli MM, Berti F, Rizzo R, Tolker-Nielsen T, Chandan V, Cescutti P
The Bep gene cluster in Burkholderia cenocepacia H111 codes for a water-insoluble exopolysaccharide essential for biofilm formation
Carbohydrate Polymers (2022) 120318
 

Burkholderia cenocepacia is an opportunistic pathogen isolated from cystic fibrosis patients where it causes infections that are extremely difficult to treat with antibiotics, and sometimes have a fatal outcome. Biofilm is a virulence trait of B. cenocepacia, and is associated with infection persistence and increased tolerance to antibiotics. In biofilms exopolysaccharides have an important role, conferring mechanical stability and antibiotic tolerance. Two different exopolysaccharides were isolated from B. cenocepacia H111 biofilms: a water-soluble polysaccharide rich in rhamnose and containing an L-Man residue, and a water-insoluble polymer made of glucose, galactose and mannose. In the present work, the product encoded by B. cenocepacia H111 bepA-L gene cluster was identified as the water-insoluble exopolysaccharide, using mutant strains and NMR spectroscopy of the purified polysaccharides. It was also demonstrated that the B. cenocepacia H111 wild type strain produces the water-insoluble exopolysaccharide in pellicles, thus underlining its potential importance in in vivo infections.

NMR spectroscopy, Exopolysaccharide structure, Biofilm, Burkholderia cenocepacia, bepA-L gene cluster

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