Vibrio cholerae RbmB is an α-1,4-polysaccharide lyase with biofilm-dispersal activity against Vibrio Polysaccharide (VPS)

Abstract

Many pathogenic bacteria form biofilms as a protective measure against environmental and host hazards. The underlying structure of the biofilm matrix consists of secreted macromolecules, usually including exopolysaccharides. To escape the biofilm, bacteria may produce a number of matrix-degrading enzymes, including glycosidic enzymes that digest exopolysaccharide scaffolds. The human pathogen Vibrio cholerae assembles and secretes an exopolysaccharide called VPS (Vibrio polysaccharide) which is essential in most cases for the formation of biofilms and consists of a repeating tetrasaccharide unit. Previous studies have indicated that a secreted glycosidase called RbmB is involved in Vibrio cholerae biofilm dispersal, although the mechanism by which this occurs is not understood. To approach the question of RbmB function, we recombinantly expressed and purified RbmB and tested its activity against purified VPS. Using a fluorescence-based biochemical assay, we show that RbmB specifically cleaves VPS in vitro under physiological conditions. Analysis of the cleavage products using mass spectrometry, solid-state NMR, and solution NMR indicates that RbmB cleaves VPS at a specific site (at the -1,4 linkage between D-galactose and a modified L-guluronic acid) into a mixture of tetramers and octamers. We demonstrate that the product of the cleavage contains a double bond in the modified-guluronic acid ring, strongly suggesting that RbmB is cleaving using a glycoside lyase mechanism. Finally, we show that recombinant RbmB from Vibrio cholerae and the related aquatic species Vibrio coralliilyticus are both able to disperse living Vibrio cholerae biofilms. Our results support the role of RbmB as a polysaccharide lyase involved in biofilm dispersal as well as an additional glycolytic enzyme to add to the toolbox of potential therapeutic antibacterial enzymes. Author summaryBiofilms are protective sheaths produced by many bacteria that play important roles in survival in the environment and in hosts during infection. Understanding how biofilms form and disperse is essential in the fight against harmful bacterial pathogens. Vibrio cholerae is a historically significant human pathogen that forms biofilms made of a polysaccharide called VPS and secreted accessory proteins. Within the V. cholerae biofilm gene cluster is a gene encoding a protein called RbmB, which is known to play a role in biofilm dispersal. Using a combination of biochemical and analytical tools, we show that RbmB indeed cleaves VPS into 4- and 8-monosaccharide fragments at a unique site and demonstrate that it falls into the family of polysaccharide lyase enzymes (as opposed to the more common hydrolases). Furthermore, recombinant RbmB disperses living V. cholerae biofilms further indicating that it may play a role in VPS processing or degradation.