Pseudomonas aeruginosa Leucine Aminopeptidase Influences Early Biofilm Composition and Structure via Vesicle-Associated Antibiofilm Activity.
Pseudomonas aeruginosa, known as one of the leading causes of disease in cystic fibrosis (CF) patients, secretes a variety of proteases. These enzymes contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds, and chronic wounds. We previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known to contribute to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. We hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm microcolony composition to increase cellular density, while decreasing matrix polysaccharide content, and that OMVs from PaAP-expressing strains but not PaAP alone or in combination with PaAP deletion strain-derived OMVs could complement this phenotype. We additionally found that OMVs from PaAP-expressing strains could cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. Finally, we showed that the OMVs could also mediate the detachment of biofilms formed by both nonself P. aeruginosa strains and Klebsiella pneumoniae, another respiratory pathogen. Our findings represent novel roles for OMVs and the aminopeptidase in the modulation of P. aeruginosa biofilm architecture.IMPORTANCE Biofilm formation by the bacterial pathogen P. aeruginosa is known to contribute to drug resistance in nosocomial infections and chronic lung infections of cystic fibrosis patients. In order to treat these infections more successfully, the mechanisms of bacterial biofilm development must be elucidated. While both bacterially secreted aminopeptidase and outer membrane vesicles have been shown to be abundant in P. aeruginosa biofilm matrices, the contributions of each of these factors to the steps in biofilm generation have not been well studied. This work provides new insight into how these bacterial components mediate the formation of a robust, drug-resistant extracellular matrix and implicates outer membrane vesicles as active components of biofilm architecture, expanding our overall understanding of P. aeruginosa biofilm biology.
['Esoda|Caitlin N|CN|', 'Kuehn|Meta J|MJ|0000-0003-0519-3019']
D018441:Biofilms D003550:Cystic Fibrosis D004847:Epithelial Cells D000067128:Extracellular Vesicles D017353:Gene Deletion D054884:Host-Pathogen Interactions D006801:Humans D007711:Klebsiella pneumoniae D007931:Leucyl Aminopeptidase D011480:Protease Inhibitors D011552:Pseudomonas Infections D011550:Pseudomonas aeruginosa
cystic fibrosis OMV Gram-negative bacteria M28 metalloprotease extracellular membrane vesicle lung biofilm microbial pathogenesis