Type II Secretion-Dependent Degradative and Cytotoxic Activities Mediated by Stenotrophomonas maltophilia Serine Proteases StmPr1 and StmPr2

Abstract

Stenotrophomonas maltophilia is an emerging opportunistic pathogen that primarily causes pneumonia and bacteremia in immunocompromised individuals. We recently reported that S. maltophilia strain K279a encodes the Xps type II secretion system and that Xps promotes rounding, actin rearrangement, detachment, and death in the human lung epithelial cell line A549. Here, we show that Xps-dependent cell rounding and detachment occur with multiple human and murine cell lines and that serine protease inhibitors block Xps-mediated rounding and detachment of A549 cells. Using genetic analysis, we determined that the serine proteases StmPr1 and StmPr2, which were confirmed to be Xps substrates, are predominantly responsible for secreted proteolytic activities exhibited by strain K279a, as well as the morphological and cytotoxic effects on A549 cells. Supernatants from strain K279a also promoted the degradation of type I collagen, fibrinogen, and fibronectin in a predominantly Xps-and protease-dependent manner, although some Xps-independent degradation of fibrinogen was observed. Finally, Xps, and predominantly StmPr1, degraded interleukin 8 (IL-8) secreted by A549 cells during coculture with strain K279a. Our findings indicate that while StmPr1 and StmPr2 are predominantly responsible for A549 cell rounding, extracellular matrix protein degradation, and IL-8 degradation, additional Xps substrates also contribute to these activities. Altogether, our data provide new insight into the virulence potential of the S. maltophilia Xps type II secretion system and its StmPr1 and StmPr2 substrates. The Gram-negative bacterium Stenotrophomonas maltophilia, prevalent in the aqueous environment, is now emerging as a prominent opportunistic and nosocomial pathogen (1, 2). S. maltophilia infects an array of host tissues and organs, including the respiratory tract, blood, bone, soft tissue, eye, urinary tract, heart, and brain. However, pneumonia is the most common infection associated with S. maltophilia, followed by bloodstream infection as the second most common (1, 2). S. maltophilia, though relatively nonvirulent for healthy individuals, can cause serious infection in immunocompromised individuals. Therefore, S. maltophilia poses the biggest threat for patients with severe burns, cystic fibrosis, and HIV, as well as those undergoing chemotherapy or immunosuppressive therapy (3). Prolonged hospitalization in intensive care units and long-term antibiotic treatment are also considered risk factors for developing pneumonia and bacteremia, for which mortality rates can range from 23 to 77% and 14 to 69%, respectively (1, 4). Community-acquired S. maltophilia infections, especially in the high-risk populations mentioned, have been reported (5), and recently, a communityacquired skin infection was reported for the first time in an immunocompetent individual (6). The multidrug-resistant nature of S. maltophilia makes treatment of infections highly difficult (7), and in recent years, an increased resistance to the preferred antibiotic trimeth...

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

“…There have been a number of virulence-related genes identified for S. maltophilia K279a, particularly those secreted through the type II-Xps system including the StmPr1-Pr3 proteases (27,29,63). In response to SCFM2, only stmPr2 is differentially expressed (decreased 9.6-fold), and we also note that all of the strains slightly increase the Sec translocon genes and strongly downregulate the Gsp system genes.…”

“…This substance, which is expectorated as sputum, serves as the primary nutrient source for pathogens growing in the CF respiratory tract and is known to influence the virulence of P. aeruginosa, B. cenocepacia, and Mycobacterium abscessus (34,38,39). These conditions are substantially different than medium conditions used to investigate S. maltophilia virulence to date (29,(49)(50)(51)(52). Here, we characterized the transcriptional responses of three S. maltophilia strains during exposure to synthetic cystic fibrosis sputum medium (SCFM2), with the eventual goal of identifying genes and metabolic pathways within the core S. maltophilia genome that contribute to S. maltophilia fitness in the CF lung, as was done previously with P. aeruginosa (40).…”

“…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.…”

Global and local selection acting on the pathogen Stenotrophomonas maltophilia in the human lung

“…Many of these lineage-separating mutations appear in genes known to be important for virulence (Supplementary Table 2): for example, one mutation in a secreted serine protease (StmPr2/AprV2 homologue), recently shown to contribute to toxicity in human epithelial cells as well as degradation of extracellular matrix protein and interleukin-8 (ref. 23), could disrupt a stabilizing disulfide bridge in a disordered region of the protein (Supplementary Fig. 5).…”

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].…”