Disruption of the Endothelial Barrier by Proteases from the Bacterial Pathogen Pseudomonas aeruginosa: Implication of Matrilysis and Receptor Cleavage

Abstract

Within the vasculature, uncontrolled pericellular proteolysis can lead to disruption of cell-to-cell and cell-to-matrix interactions and subsequent detachment-induced cell apoptosis, or anoikis, contributing to inflammatory vascular diseases, with the endothelium as the major target. Most studies so far have focused on endogenous proteinases. However, during bloodstream infections, bacterial proteinases may also trigger endothelial anoikis. We thus investigated the potential apoptotic activity of the proteinases secreted by the haematotropic opportunistic pathogen, Pseudomonas aeruginosa, and particularly its predominant metalloproteinase, LasB. For this, we used the secretome of the LasB-expressing pseudomonal strain, PAO1, and compared it with that from the isogenic, LasB-deficient strain (PAO1∆lasB), as well as with purified LasB. Secretomes were tested for apoptotic activity on cultured human endothelial cells derived from the umbilical vein or from the cerebral microvasculature. We found that the PAO1 secretome readily induced endothelial cell anoikis, as did secretomes of LasB-positive clinical pseudomonal isolates, while the PAO1∆lasB secretome had only a limited impact on endothelial adherence and viability. Notably, purified LasB reproduced most of the effects of the LasB-containing secretomes, and these were drastically reduced in the presence of the LasB-selective inhibitor, phosphoramidon. A precocious and extensive LasB-dependent degradation of several proteins associated with the endothelial extracellular matrix, fibronectin and von Willebrand factor, was observed by immunofluorescence and/or immunoblotting analysis of cell cultures. Moreover, the PAO1 secretome, but not that from PAO1∆lasB, specifically induced rapid endoproteolysis of two major interendothelial junction components, VE-cadherin and occludin, as well as of the anti-anoikis, integrin-associated urokinase receptor, uPAR. Taken as a prototype for exogenous haemorrhagic proteinases, pseudomonal LasB thus appears to induce endothelial anoikis not only via matrilysis, as observed for many pro-apoptotic proteinases, but also via cleavage of some essential cell-to-cell and cell-to-matrix adhesion receptors implicated in the maintenance of the endothelial barrier.

The therapeutic potential of the insect metalloproteinase inhibitor against infections caused by Pseudomonas aeruginosa

“…IMPI‐GST showed no toxicity towards HaCaT cells in the concentration range 20 n m to 60 μ m . In contrast, Sec caused a strong and concentration‐dependent decrease in viability (Figure ) and the effect was stronger than reported in earlier studies, which did not report the total loss of cells following a 8 h incubation with 10% Sec. The higher PE concentration in our Sec preparation (0.944 ± 0.07 μ m ) may explain this phenomenon.…”

“…As the concentration of Sec increased, F‐actin became less abundant and the remaining fibers retracted to the cell nucleus and became indistinct as the cell monolayer became more porous. These structures are recognizable in cell images presented in other studies following exposure to PE or Sec . Several proteins (e.g.…”

“…In P. aeruginosa , one of the key virulence factors is a M4 metalloproteinase known as Pseudomonas elastase (PE), LasB or pseudolysin, which acts on a wide range of substrates . Other proteases, such as alkaline protease and serine protease IV, are thought to have little or no proteolytic activity . PE confers the toxicity of P. aeruginosa and also facilitates invasion and immunomodulation .…”

“…PE also inactivates components of the host immune system and induces the production of interleukin 8 (IL‐8) . In airway epithelial cells, PE increases the permeability of monolayers, induces anoikis (a form of programmed cell death triggered by detachment) and disrupts tight and adherens junctions . PE also degrades filamentous actin (F‐actin), thus affecting cell layer permeability and cell migration .…”

Urokinase receptor-deficient mice mount an innate immune response to and clarify respiratory viruses as efficiently as wild-type mice

“…For example, the metalloproteinase LasB of Pseudomonas aeruginosa induces the endoproteolysis of uPAR, a process which appears to correlate with disruption of the endothelial barrier. 19 Also, uPA contributes to plasmin recruitment on the cell surface of Group A Streptococcus, thereby favoring bacterial invasiveness. 20 In conclusion, uPAR plays distinct roles in bacterial and viral respiratory infections.…”