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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

NCBI PubMed ID: 32131450
Publication DOI: 10.3390/ijms21051702
Journal NLM ID: 101092791
Publisher: Basel, Switzerland: MDPI
Correspondence: pcescutti@units.it
Institutions: Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa, Department of Life Sciences, University of Trieste, via L. Giorgieri 1, Bdg C11, 34127 Trieste, Italy, Department of Food Science, Cornell University, Ithaca, NY 14853, USA, Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen, DK-2200 Copenhagen, Denmark
Methods: 13C NMR, 1H NMR, GLC-MS, NMR-2D, sugar analysis, GLC, MD simulations

Burkholderia cenocepacia belongs to the Burkholderia Cepacia Complex, a group of 22 closely related species both of clinical and environmental origin, infecting cystic fibrosis patients. B. cenocepacia accounts for the majority of the clinical isolates, comprising the most virulent and transmissible strains. The capacity to form bioﬁlms is among the many virulence determinants of B. cenocepacia, a characteristic that confers enhanced tolerance to some antibiotics, desiccation, oxidizing agents, and host defenses. Exopolysaccharides are a major component of bioﬁlm matrices, particularly providing mechanical stability to bioﬁlms. Recently, a water-insoluble exopolysaccharide produced by B. cenocepacia H111 in biofilm was characterized. In the present study, a water-soluble exopolysaccharide was extracted from B. cenocepacia H111 biofilm, and its structure was determined by GLC-MS, NMR and ESI-MS. The repeating unit is a linear rhamno-tetrasaccharide with 50% replacement of a 3-α-L-Rha with a α-3-L-Man. [2)-α-L-Rhap-(1→3)-α-L-[Rhap or Manp]-(1→3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→]n Molecular modelling was used to obtain information about local structural motifs which could give information about the polysaccharide conformation.

NMR, molecular modelling, polysaccharide structure, Biofilm, Burkholderia cenocepacia H111, L-mannose

NCBI PubMed ID: 33440288
Publication DOI: 10.1016/j.carres.2020.108231
Journal NLM ID: 0043535
Publisher: Elsevier
Correspondence: Paola Cescutti
Institutions: Department of Chemistry, University of Cape Town, Rondebosch, 7701, South Africa, Department of Life Sciences, University of Trieste, Via L. Giorgieri 1, Bdg. C11, 34127, Trieste, Italy, Department of Food Science, Cornell University, Ithaca, NY, 14853, USA, Costerton Biofilm Center, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200, Copenhagen, Denmark
Methods: 13C NMR, 1H NMR, GLC-MS, NMR-2D, partial acid hydrolysis, ESI-MS, GLC, MD simulations, composition analysis

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

NCBI PubMed ID: 36436859
Publication DOI: 10.1016/j.carbpol.2022.120318
Journal NLM ID: 8307156
Publisher: Elsevier
Correspondence: pcescutti@units.it
Institutions: Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen, DK-2200 Copenhagen, Denmark, GSK, Via Fiorentina 1, 53100 Siena, Italy, Department of Life Sciences, University of Trieste, via L. Giorgieri 1, Bdg. C11, 34127 Trieste, Italy
Methods: 1H NMR, sugar analysis, GLC, HR-MAS NMR, pellicles production, sequence analysis of gene clusters

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