Pseudomonas aeruginosa Exoprotein-Induced Barrier Disruption Correlates With Elastase Activity and Marks Chronic Rhinosinusitis Severity.

Sarah Vreugde,Alkis J Psaltis,Mahnaz Ramezanpour,Stephanie A Fong,Jae Murphy,Masanobu Suzuki,Peter John Wormald,Jian Li,Clare Cooksley

doi:10.3389/fcimb.2019.00038

Abstract

Background: Pseudomonas aeruginosa causes severe chronic respiratory diseases and is associated with recalcitrant chronic rhinosinusitis (CRS). P. aeruginosa exoproteins contain virulence factors and play important roles in the pathogenicity of P. aeruginosa, however their role in CRS pathophysiology remains unknown.Methods: We isolated P. aeruginosa clinical isolates (CIs) and obtained clinical information from 21 CRS patients. Elastase activity of the CIs was measured at different phases of growth. Primary human nasal epithelial cells (HNECs) were cultured at air-liquid interface (ALI) and challenged with P. aeruginosa exoproteins or purified elastase, followed by measuring Transepithelial Electrical Resistance (TEER), permeability of FITC-dextrans, western blot, and immunofluorescence.Results: 14/21 CIs had a significant increase in elastase activity in stationary phase of growth. There was a highly significant strong correlation between the in vitro elastase activity of P. aeruginosa CIs with mucosal barrier disruption evidenced by increased permeability of FITC-dextrans (r = 0.95, p = 0.0004) and decreased TEER (r = −0.9333, P < 0.01) after 4 h of challenge. Western blot showed a significant degradation of ZO-1, Occludin and β-actin in relation to the elastase activity of the exoproteins. There was a highly significant correlation between the in vitro elastase activity of P. aeruginosa CIs and CRS disease severity (for log phase, r = 0.5631, p = 0.0097; for stationary phase, r = 0.66, p = 0.0013) assessed by CT imaging of the paranasal sinuses.Conclusion: Our results implicate P. aeruginosa exoproteins as playing a major role in the pathophysiology of P. aeruginosa associated CRS by severely compromising mucosal barrier structure and function.

Highlights

Pseudomonas aeruginosa is an opportunistic pathogen causing a wide range of community and nosocomial infections of the airway, urinary tract, and skin such as chronic wounds and burns
To test the effect of exoproteins on the mucosal barrier in vitro, primary HNEC-Air-Liquid Interface (ALI) cultures were established from 4 independent control patients without chronic rhinosinusitis (CRS) or cystic fibrosis (CF) followed by measuring Trans Epithelial Electrical Resistance (TEER) and passage of Dextran-FITC
Exoproteins were collected from log and stationary growth phases of 4 P. aeruginosa strains [PAO1 reference strain and three clinical isolates (CIs) collected from the sinus cavities of CRS patients—one with CF (S.M.), two without], filtered and protein concentration measured (Supplementary Figure 1 and Supplementary Table 1)

Summary

Introduction

Pseudomonas aeruginosa is an opportunistic pathogen causing a wide range of community and nosocomial infections of the airway, urinary tract, and skin such as chronic wounds and burns. Secreted proteases include elastase A (LasA), elastase B (LasB), alkaline protease (AP), protease IV (PIV), and P. aeruginosa aminopeptidase (PAAP) They interact with the host during pathogenic infections and have a critical role in invasiveness (Janda and Bottone, 1981; Bleves et al, 2010). P. aeruginosa elastase (LasB) is a major exoprotein and the primary elastolytic enzyme (Peters and Galloway, 1990) It plays an important role in the pathogenesis of P. aeruginosa infection and has been shown to have a highly efficient proteolytic activity on a range of host proteins. P. aeruginosa exoproteins contain virulence factors and play important roles in the pathogenicity of P. aeruginosa, their role in CRS pathophysiology remains unknown

Methods

Results

Conclusion