Stenotrophomonas maltophilia Serine Protease StmPr1 Induces Matrilysis, Anoikis, and Protease-Activated Receptor 2 Activation in Human Lung Epithelial Cells

Abstract

is an emerging, opportunistic nosocomial pathogen that can cause severe disease in immunocompromised individuals. We recently identified the StmPr1 and StmPr2 serine proteases to be the substrates of the Xps type II secretion system in strain K279a. Here, we report that a third serine protease, StmPr3, is also secreted in an Xps-dependent manner. By constructing a panel of protease mutants in strain K279a, we were able to determine that StmPr3 contributes to the previously described Xps-mediated rounding and detachment of cells of the A549 human lung epithelial cell line as well as the Xps-mediated degradation of fibronectin, fibrinogen, and the cytokine interleukin-8 (IL-8). We also determined that StmPr1, StmPr2, and StmPr3 account for all Xps-mediated effects toward A549 cells and that StmPr1 contributes the most to Xps-mediated activities. Thus, we purified StmPr1 from the strain K279a culture supernatant and evaluated the protease's activity toward A549 cells. Our analyses revealed that purified StmPr1 behaves more similarly to subtilisin than to trypsin. We also determined that purified StmPr1 likely induces cell rounding and detachment of A549 cells by targeting cell integrin-extracellular matrix connections (matrilysis) as well as adherence and tight junction proteins for degradation. In this study, we also identified anoikis as the mechanism by which StmPr1 induces the death of A549 cells and found that StmPr1 induces A549 IL-8 secretion via activation of protease-activated receptor 2. Altogether, these results suggest that the degradative and cytotoxic activities exhibited by StmPr1 may contribute to pathogenesis in the lung by inducing tissue damage and inflammation.

Antibacterial and Antivirulence Activity of Glucocorticoid PYED-1 against Stenotrophomonas maltophilia

“…In addition, PYED-1 significantly decreased the expression of sphB, StmPr1, and StmPr3 genes encoding for serine proteases that contribute to degradation of extracellular matrix proteins [38]. The ability of S. maltophilia to produce extracellular protease may contribute to S. maltophilia pathogenesis in the lungs of CF patients [39,40]. The major protease StmPr1 induces the death of A549 fibroblasts and IL-8 secretion by A549 cells [40].…”

“…The ability of S. maltophilia to produce extracellular protease may contribute to S. maltophilia pathogenesis in the lungs of CF patients [39,40]. The major protease StmPr1 induces the death of A549 fibroblasts and IL-8 secretion by A549 cells [40]. PYED-1 treatment could reduce the release of extracellular proteases, and consequently tissue damage and inflammation in the host.…”

Stenotrophomonas maltophilia Encodes a VirB/VirD4 Type IV Secretion System That Modulates Apoptosis in Human Cells and Promotes Competition against Heterologous Bacteria, Including Pseudomonas aeruginosa

“…Mutation of the VirB/D4 T4SS of S. maltophilia. To begin to determine the roles of T4SS in S. maltophilia physiology and pathogenesis, we introduced an unmarked deletion into the virB10 gene (i.e., RS14310), using methods that we previously employed to construct a variety of strain K279a mutants (27)(28)(29)(30). We continued to use K279a as the parental strain for our mutational analyses, because it is representative of contemporary virulent strains of S. maltophilia (83,84).…”

“…The A549 cell line is widely used for the study of lung pathogens, including S. maltophilia (86)(87)(88)(89)(90). We previously linked the death of the A549 cells to an induction of apoptosis (i.e., caspase-3/7 activation) that is largely due to a protease released by the T2SS (28)(29)(30). Such detachment-induced apoptosis is referred to as anoikis (30).…”

Stenotrophomonas maltophilia Differential Gene Expression in Synthetic Cystic Fibrosis Sputum Reveals Shared and Cystic Fibrosis Strain-Specific Responses to the Sputum Environment

“…While these studies have provided valuable insight into S. maltophilia adaptation to the CF lung, the molecular mechanisms permitting lung colonization and virulence remain largely unknown. Invertebrate infection models, bioinformatics, and tissue culture-based approaches have been useful in identifying virulence-associated genes, including those for a variety of secreted proteases and hydrolytic enzymes, adhesins, and siderophores (26)(27)(28)(29)(30)(31)(32). However, it is important to note that many of these studies were performed under conditions that do not reflect the environment of the CF lung.…”