Identifying the Protein(s) in Lung Secretions that Inhibit P. aeruginosa Swimming Motility

Background: In human lung secretions there are many anti-microbial proteins that help prevent bacterial colonization. However, lung secretions from patients with cystic fibrosis (CF) show reductions in antimicrobial activity. Previous studies in our lab demonstrated that wild-type, but not CF-like, airway secretions inhibited P. aeruginosa swimming motility in vitro. P. aeruginosa is the leading cause of death in CF and its swimming motility is associated with increased virulence. Subsequent preliminary experiments suggested that this inhibition is mediated by a protein between 50 and 250 kDa in size.  The goal of this research is to identify the protein(s) responsible for inhibition P. aeruginosaswimming motility which is active in non-CF airway secretions but is less active or inactive in CF-like airway secretions. Methods: Swimming of the P. aeruginosaPA14 strain was quantitated using microscopic and computer analysis. Apical secretions from Calu-3 cells (wild-type or CF-like) grown in air-interface monolayers was collected in PBS and used to pre-treat PA14 cultures before and during swimming analysis. Size-exclusion spin columns and gel electrophoresis were used to separate proteins in wild-type and CF-like apical secretions for analysis and use in the swimming assay. Results:Boiling and fractionation of wild-type secretions removed the inhibition activity in wild-type secretions. Gel electrophoresis of both wild-type Calu-3 secretions showed multiple protein bands between 50 and 250 kDa. Conclusions: Comparison of gel electrophoresis results with the published proteome of apical airway secretions limits the potential candidates present in wild-type Calu-3 secretions to less than 15 known proteins, some with known or suspected anti-microbial activity.