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

Ahmad Aljohmani,Bastian Opitz,Daniela Yildiz,Markus Bischoff

doi:10.3390/ijms23031259

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.

Highlights

Pneumonia is a major complication that often requires hospitalization and is highly associated with a substantial increase in morbidity and mortality [1]
Our present study shows that epithelial ADAM10 is involved in the inflammatory manifestations in response to P. aeruginosa infection and Exotoxin A (ExoA) stimulation by modulating transepithelial leukocyte migration, protein permeability and epithelial regeneration
No influence by S. pneumoniae was observed. This pathogen-specific regulation of ADAM10 might be predominantly depending on the secreted toxin repertoire of the pathogen and the induction of calcium influx in the case of ExoA rather than interaction with the bacterial particle itself

Summary

Introduction

Pneumonia is a major complication that often requires hospitalization and is highly associated with a substantial increase in morbidity and mortality [1]. Streptococcus pneumoniae (S. pneumoniae) and Pseudomonas aeruginosa (P. aeruginosa) are the most common causes of Gram-positive community acquired pneumonia and Gram-negative hospital acquired pneumonia, respectively [2,3] These bacterial pathogens, which are frequently found as colonizers of the upper airways, and which can reach the lower respiratory tract by aspiration, can cause pneumonia if the pulmonary immune system is not immediately able of clearing the infection. Epithelial cells do form the first mechanical barrier against invading pathogens, but further function as “non-traditional immune cells” [4] Their activation can be initiated either through interaction with the pathogens via Toll-like receptors (TLRs) and other pattern recognition receptors located on the cell surface, or through intracellular sensing of bacterial derivatives like DNA, cell wall fragment or the secreted toxins [5,6]. P. aeruginosa, as one example, is recognized by the lung epithelium through TLR2 and TLR4, thereby initiating a proinflammatory response and secretion of proinflammatory cytokines such as IL-1β IL-8, IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) [7–10]

Objectives

Methods

Results

Conclusion