Aminopeptidase-Dependent Modulation of Bacterial Biofilms by Pseudomonas aeruginosa Outer Membrane Vesicles

Abstract

Pseudomonas aeruginosa, known as one of the leading causes of morbidity and mortality in cystic fibrosis (CF) patients, secretes a variety of virulence-associated proteases. These enzymes have been shown to 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. Our lab has previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is also highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known for their contribution to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. With this in mind, 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 resistance to the antibiotic colistin. We recreate this phenotype using a pellicle biofilm model, in which bacteria are grown statically at the culture air-liquid interface, demonstrating that these phenotypes are not dependent on the coculture host cell substrate. We additionally show that OMVs from PaAP expressing strains, but not PaAP alone or in combination with PaAP deletion strain-derived OMVs, could complement this phenotype. Finally, we found that OMVs from PaAP-expressing strains cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. OMVs mediated the detachment of biofilms formed by both non-self P. aeruginosa strains and K. pneumoniae, another respiratory pathogen, showing that this process may also be relevant in polymicrobial communities and acts on non-P. aeruginosa derived substrates. Our findings represent novel roles for OMVs and the PaAP aminopeptidase in the modulation of bacterial biofilm architecture.