Impaired Airway Epithelial Barrier Integrity in Response to Stenotrophomonas maltophilia Proteases, Novel Insights Using Cystic Fibrosis Bronchial Epithelial Cell Secretomics.

Kevin Molloy,Kieran Wynne,Noel G Mcelvaney,Gerard Cagney,Catherine M Greene,Eugene T Dillon

doi:10.3389/fimmu.2020.00198

Abstract

Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can chronically colonize the lungs of people with cystic fibrosis (CF) and is associated with lethal pulmonary hemorrhage in immunocompromised patients. Its secreted virulence factors include the extracellular serine proteases StmPR1, StmPR2, and StmPR3. To explore the impact of secreted virulence determinants on pulmonary mucosal defenses in CF, we examined the secretome of human CFBE41o- bronchial epithelial cells in response to treatment with S. maltophilia K279a cell culture supernatant (CS) using a liquid-chromatography-tandem mass spectrometry (LC-MS/MS) based label-free quantitative (LFQ) shotgun proteomics approach for global profiling of the cell secretome. Secretome analysis identified upregulated pathways mainly relating to biological adhesion and epithelial cell signaling in infection, whereas no specific pathways relating to the immune response were enriched. Further exploration of the potentially harmful effects of K279a CS on CF bronchial epithelial cells, demonstrated that K279a CS caused CFBE41o- cell condensation and detachment, reversible by the serine protease inhibitor PMSF. K279a CS also decreased trans-epithelial electrical resistance in CFBE41o- cell monolayers suggestive of disruption of tight junction complexes (TJC). This finding was corroborated by an observed increase in fluorescein isothiocyanate (FITC) dextran permeability and by demonstrating PMSF-sensitive degradation of the tight junction proteins ZO-1 and occludin, but not JAM-A or claudin-1. These observations demonstrating destruction of the CFBE41o- TJC provide a novel insight regarding the virulence of S. maltophilia and may explain the possible injurious effects of this bacterium on the CF bronchial epithelium and the pathogenic mechanism leading to lethal pulmonary hemorrhage.

Highlights

Culture supernatant (CS) from bacteria, most notably Pseudomonas aeruginosa, has been used extensively to study host-pathogen interactions in the cystic fibrosis (CF) lung
As a first step toward assessing the role, if any, of secreted proteases in the pathogenesis of S. maltophilia pulmonary infection, we examined the effect of differing concentrations of K279a CS (5 and 10% v/v) on CFBE41o- cell monolayers compared with untreated control cells (Figure 1A)
Using the seed proteins identified from this study we identified candidate disease proteins to determine the effect of S. maltophilia on CF bronchial epithelial cells using PPI network (PPIN) analysis from three prioritization tools: NetworkAnalyst, GeneMANIA, and ToppGene

Summary

Introduction

Culture supernatant (CS) from bacteria, most notably Pseudomonas aeruginosa, has been used extensively to study host-pathogen interactions in the cystic fibrosis (CF) lung. Studies have explored its effects on TLR-induced inflammation [1], pro-inflammatory cytokine production [2, 3], innate immunity proteins [4,5,6] and degradation of extracellular matrix components [5, 7, 8], amongst others. There is a paucity of information regarding the effects of the important emerging CF pathogen, Stenotrophomonas maltophilia on airway epithelial cells in vitro. Given that the pathogenesis of S. maltophilia is complex and multifactorial, high-throughput technologies such as proteomics can help decipher differences in protein expression in composite circumstances such as host-pathogen interactions. Proteins secreted by a pathogen are present at the interface between the pathogen and the host cells and can regulate or mediate the host responses and cause disease [9]

Methods

Results

Conclusion