Vibrio cholerae RbmB is an α-1,4-polysaccharide lyase with biofilm-disrupting activity against Vibrio polysaccharide (VPS)
by Ranjuna Weerasekera, Alexis Moreau, Xin Huang, Kee-Myoung Nam, Alex Hinbest, Yun Huynh, Xinyu Liu, Christopher Ashwood, Lauren E. Pepi, Eric Paulson, Lynette Cegelski, Jing Yan, Rich Olson
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, often 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 V. 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 process using mass spectrometry, solid-state NMR, and solution NMR indicates that RbmB cleaves VPS at a specific site (at the α-1,4 linkage between D-galactose and a modified L-gulose) 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 V. cholerae and the related aquatic species Vibrio coralliilyticus are both able to disrupt living V. 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.