Glycoside hydrolase processing of the Pel polysaccharide alters biofilm biomechanics and Pseudomonas aeruginosa virulence

Pel exopolysaccharide biosynthetic loci are phylogenetically widespread biofilm matrix determinants in bacteria. In Pseudomonas aeruginosa, Pel is crucial for cell-to-cell interactions and reducing susceptibility to antibiotic and mucolytic treatments. While genes encoding glycoside hydrolases have long been linked to biofilm exopolysaccharide biosynthesis, their physiological role in biofilm development is unclear. Here we demonstrate that the glycoside hydrolase activity of P. aeruginosa PelA decreases adherent biofilm biomass and is responsible for generating the low molecular weight secreted form of the Pel exopolysaccharide. We show that the generation of secreted Pel contributes to the biomechanical properties of the biofilm and decreases the virulence of P. aeruginosa in Caenorhabditis elegans and Drosophila melanogaster. Our results reveal that glycoside hydrolases found in exopolysaccharide biosynthetic systems can help shape the soft matter attributes of a biofilm and propose that secreted matrix components be referred to as matrix associated to better reflect their influence. Canadian Institutes of Health Research (CIHR) [MOP 43998, FDN154327]; Tier I Canada Research Chair; Tier II Canada Research Chair from CIHR; Natural Sciences and Engineering Research Council (NSERC) of Canada; Cystic Fibrosis Canada; Natural Science and Engineering Research Council of Canada [522691522691, RGPIN-2019-04119]; K99 Pathway to Independence Award [5K99GM134121-02]; Cystic Fibrosis Foundation [REICHH19F5]; American Heart Association Career Development Award [19CDA34630005] Published version The authors thank Ira Lacdao and Piyanka Sivarajah for providing recombinantly expressed and purified PelAhPa (PelA 47-303), Dr. K.G. Iliadi for help with Drosophila lifespan and statistical analyses, and Andreea A. Gheorghita for helpful discussions. We thank the following sources of funding: P. Lynne Howell: Canadian Institutes of Health Research (CIHR) MOP 43998 and FDN154327. PLH was the recipient of a Tier I Canada Research Chair (2006-2020). JJH is supported by a Tier II Canada Research Chair from CIHR (2013-2023) and a Discovery Grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada. This research has been supported by graduate scholarships from Cystic Fibrosis Canada (GBW) and the Natural Science and Engineering Research Council of Canada (GBW). CR was supported by a K99 Pathway to Independence Award (5K99GM134121-02) and a Postdoc-to-Faculty Transition Award from the Cystic Fibrosis Foundation (REICHH19F5). C. elegans work by ARW and AWR was supported by the Natural Sciences and Engineering Research Council of Canada (Grant #522691522691). Drosophila work by OBG and GLB was supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2019-04119). Erin S. Gloag was funded by an American Heart Association Career Development Award (19CDA34630005).