Exopolysaccharides Isolated from Biofilms Produced by the Pathogen Burkholderia Cenocepacia

Session: 
PS1 Poster session 1 Odd numbers
Code: 
P33
Location (hall): 
Foyer
Start/end time: 
Monday, July 1, 2019 - 15:45 to 17:15
Paola
Cescutti

Paola Cescutti1, Barbara Bellich1, Neil Ravenscroft2, Stefano Morasso1, Mustafa Fazli3, Tim Tolker-Nielsen4, Roberto Rizzo1

1Department of Life Sciences, University of Trieste, Trieste, Italy, 2Department of Chemistry, University of Cape Town, Cape Town, South Africa, 3Department of Biology, University of Copenhagen, Copenhagen, Denmark, 4Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark

Burkholderia cenocepacia is an important pathogen for cystic fibrosis patients, accounting for the majority of the clinical Burkholderia spp. isolates, and comprising the most virulent and transmissible strains, often associated with poor or lethal clinical outcome. Among the known virulence factors, the ability to form biofilms is connected with the production of exopolysaccharides (EPOLs) which are mainly involved in the mechanical stability of the biofilm matrix.

Regarding B. cenocepacia H111, a Bcam1349/c-di-GMP regulated exopolysaccharide 12-gene cluster (Bcam1330–Bcam1341) has been identified and found to be essential for biofilm formation [1]. The 12-gene cluster products appeared to be involved in the biosynthesis of a major EPOL that confers structural stability to the biofilms. Overproduction of Bcam1349 in trans promoted wrinkly colony morphology, pellicle, and biofilm formation in B. cenocepacia H111. In order to identify this major EPOL in the biofilm of B. cenocepacia H111, a strain overexpressing Bcam1349 was used to form both pellicles, a biofilm which forms at the air/liquid interface, and biofilm on agar plates. EPOLs extracted with NaOH from the biofilm matrix provided two major polymers: a water soluble EPOL containing rhamnose and mannose residues in the molar ratio 7:1 and a water insoluble EPOL made up of Gal:Glc:Man in the molar ratios 2:1:1. These EPOLs were found in the pellicles as well as in the biofilm developed on agar plates. In order to identify the EPOL biosynthesised by the 12-gene cluster coded proteins, strains with transposon insertion in two different genes of the cluster were investigated. They were not able to produce pellicles, nor wrinkly colonies on agar plates. Isolation and NMR analysis of the EPOLs produced by these two strains revealed the absence of the water-insoluble EPOL, strongly associating it to the wrinkly phenotype and indicating it as the product of the 12-gene cluster coded proteins.

Investigation of the insoluble EPOL structure by GLC-MS and NMR spectroscopy suggested the following tetrasaccharide repeating unit:

[3)-α-D-Gal-(1→3)-α-D-Man-(1→3)-α-D-Gal-(1→3)-α-D-Glc-(1→]n

Its insolubility may well account for the wrinkly phenotype and the pellicle formation.

References: 
  1. Fazli, M.; McCarthy, Y.; Givskov, M.; Ryan, R.P.; Tolker-Nielsen T. The exopolysaccharide gene cluster Bcam1330–Bcam1341 is involved in Burkholderia cenocepacia biofilm formation, and its expression is regulated by c-di-GMP and Bcam1349. MicrobiologyOpen 2013, 2(1), 105–122.
Weight: 
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