Cellulophaga algicola alginate lyase and Pseudomonas aeruginosa Psl glycoside hydrolase inhibit biofilm formation by Pseudomonas aeruginosa CF2843 on three-dimensional aggregates of lung epithelial cells

<i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that produces a biofilm containing the polysaccharides, alginate, Psl, and Pel, and causes chronic lung infection in cystic fibrosis patients. Others and we have previously explored the use of alginate lyases in inhibiting <i>P. aeruginosa</i> biofilm formation on plastic and lung epithelial cell monolayers. We now employ a more physiologically representative model system, i.e., three-dimensional aggregates of A549 lung epithelial cells cultured under conditions of microgravity in a rotary cell culture system to mimic the natural lung environment, and a previously isolated clinical strain, <i>Pseudomonas aeruginosa</i> CF2843 that we engineered by transposon-mediated integration to express Green Fluorescent Protein and for which we also report the complete genome sequence. Immunostaining and lectin binding studies indicated that the three-dimensional cell aggregates harbored sialylated and fucosylated epitopes as well as Muc1, Muc5Ac, and β-catenin on their surfaces, suggestive of mucin secretion and the presence of tight junctions, hallmark features of lung epithelial tissue. Using this validated model system with confocal microscopy and viable bacterial counts as readouts, we demonstrated that <i>Cellulophaga algicola</i> alginate lyase and <i>Pseudomonas aeruginosa</i> Psl glycoside hydrolase, but not <i>Pseudomonas aeruginosa</i> Pel glycoside hydrolase, inhibit biofilm formation by <i>Pseudomonas aeruginosa</i> on three-dimensional lung epithelial cell aggregates.