Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans

Abstract

Pneumonia is a life-threatening disease often caused by infection with Streptococcus pneumoniae and Pseudomonas aeruginosa. Many of the mediators (e.g., TNF, IL-6R) and junction molecules (e.g., E-cadherin) orchestrating inflammatory cell recruitment and loss of barrier integrity are proteolytically cleaved through a disintegrin and metalloproteinases (ADAMs). We could show by Western blot, surface expression analysis and measurement of proteolytic activity in cell-based assays, that ADAM10 in epithelial cells is upregulated and activated upon infection with Pseudomonas aeruginosa and Exotoxin A (ExoA), but not upon infection with Streptococcus pneumoniae. Targeting ADAM10 by pharmacological inhibition or gene silencing, we demonstrated that this activation was critical for cleavage of E-cadherin and modulated permeability and epithelial integrity. Stimulation with heat-inactivated bacteria revealed that the activation was based on the toxin repertoire rather than the interaction with the bacterial particle itself. Furthermore, calcium imaging experiments showed that the ExoA action was based on the induction of calcium influx. Investigating the extracellular vesicles and their proteolytic activity, we could show that Pseudomonas aeruginosa triggered exosomal release of ADAM10 and proteolytic cleavage in trans. This newly described mechanism could constitute an essential mechanism causing systemic inflammation in patients suffering from Pseudomonas aeruginosa-induced pneumonia stimulating future translational studies.

Pseudomonas aeruginosa Alters Critical Lung Epithelial Cell Functions through Activation of ADAM17

“…Pseudomonas aeruginosa ( P. aeruginosa , PA103 strain) and Streptococcus pneumoniae ( S. pneumoniae , R6 strain) were grown as described earlier [ 12 ]. Briefly, the bacteria were incubated in Todd–Hewitt–Bouillon (THB) medium at 37 °C at 150 rpm until reaching the exponential phase.…”

“…In each experiment (as indicated in the figure legends) the cells were pre-incubated with TAPI-1 (10 µM) or DMSO (0.1%) for 30 min. Subsequently, the cells were infected with either P. aeruginosa or S. pneumoniae or stimulated with Exotoxin A (ExoA, Merck Millipore, Darmstadt, Germany) as indicated in the figure legends [ 12 , 17 , 20 ].…”

“…Western blot analysis was carried out as described [ 12 ]. Cell lysates were obtained by incubating the cells with lysis buffer (20 mM Tris·HCl, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, 10 mM 1,10- phenanthroline monohydrate, 1x Complete inhibitor), and protein concentration was determined using the bicinchoninic acid assay (BCA) kit from Thermo Fisher (Karlsruhe, Germany).…”

“…Lentivirus production and cell transduction were performed as described earlier [ 12 ]. The production of lentivirus was performed by transfecting HEK293T cells as described [ 20 ].…”

“…ExoA-producing strains show a much higher virulence compared to ExoA-deficient mutants, which can be prevented by vaccination with inactivated ExoA [ 11 ]. This detrimental action is at least in parts based on lung epithelial barrier disruption, which we could link to activation of a disintegrin and metalloproteinase (ADAM) 10 in a recent study [ 12 , 13 ].…”