Production of Outer Membrane Vesicles by the Plague Pathogen Yersinia pestis

Lathem WW (2014) Production of Outer Membrane Vesicles by the Plague Pathogen Yersinia pestis. PLoS ONE 9(9): e107002. doi:10.1371/journal.pone.0107002 Production of Outer Membrane Vesicles by the Plague Pathogen Yersinia pestis Justin L. Eddy. 0 Lindsay M. Gielda. 0 Adam J. Caulfield 0 Stephanie M. Rangel 0 Wyndham W. Lathem 0 Lisa A. Morici, Tulane University School of Medicine, United States of America 0 Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine , Chicago, Illinois , United States of America Many Gram-negative bacteria produce outer membrane vesicles (OMVs) during cell growth and division, and some bacterial pathogens deliver virulence factors to the host via the release of OMVs during infection. Here we show that Yersinia pestis, the causative agent of the disease plague, produces and releases native OMVs under physiological conditions. These OMVs, approximately 100 nm in diameter, contain multiple virulence-associated outer membrane proteins including the adhesin Ail, the F1 outer fimbrial antigen, and the protease Pla. We found that OMVs released by Y. pestis contain catalytically active Pla that is competent for plasminogen activation and a2-antiplasmin degradation. The abundance of OMV-associated proteins released by Y. pestis is significantly elevated at 37uC compared to 26uC and is increased in response to membrane stress and mutations in RseA, Hfq, and the major Braun lipoprotein (Lpp). In addition, we show that Y. pestis OMVs are able to bind to components of the extracellular matrix such as fibronectin and laminin. These data suggest that Y. pestis may produce OMVs during mammalian infection and we propose that dispersal of Pla via OMV release may influence the outcome of infection through interactions with Pla substrates such as plasminogen and Fas ligand. - Funding: National Institutes of Health P30 CA060553 to RHLCCC; National Institutes of Health R01 AI093727 to WWL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Outer membrane vesicles (OMVs) are closed spherical portions of the bacterial outer membrane that contain phospholipids, outer membrane proteins, lipopolysacharide (LPS), and periplasmic contents [1]. Produced by many Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Helicobacter pylori [24], OMVs are formed when small portions of the outer membrane pinch off from the cell and are released as selfcontained spherical structures that range from 20250 nm in size [5]. While the biogenesis of OMVs is poorly understood, it is thought that expansion of the outer leaflet of the membrane relative to the inner leaflet induces membrane curvature that forces the outer membrane to bud away from the cell [5,6]. OMV production can be detected in bacterial communities growing under a variety of conditions, including planktonic cultures as well as in surface-attached biofilm communities [7,8]. OMVs are produced by both pathogenic and non-pathogenic bacteria [911]. OMVs released by pathogens can contain multiple components that interact with the host, including LPS, virulence factors, and other antigens. Pathogen-derived OMVs may contribute to virulence by modulating the innate immune response, delivering toxins to cells, dispersing antigens and virulence factors away from the bacterium, trafficking signaling molecules between bacteria, and more. Microscopic examination of tissues has detected the presence of OMVs near host cells or within host tissues, suggesting an interaction between OMVs and the host during infection [1214]. Further, OMVs have been found to deliver active toxins to host cells, including the enterotoxigenic E. coli heat-labile enterotoxin (LT), the enterohemorrhagic E.coli pore-forming cytotoxin ClyA, and the H. pylori VacA protein [3,11,15]. Environmental stresses contribute to the production of OMVs [16], suggesting that, as bacteria encounter stressors such as those found within the infected host, the production of OMVs may not only manipulate interactions with the host but also aid in the survival of the bacterium. The Gram-negative bacterium Yersinia pestis, a pathogen of both insects and mammals, can be transmitted to humans via the bite of hematophagous insects (typically fleas) or through the inhalation of respiratory droplets or aerosols containing the bacteria, and can cause bubonic, pneumonic, or septicemic plague [17]. Temperature is a major regulator of gene expression in Y. pestis, controlling both transcriptional and post-transcriptional responses [18,19]. At lower temperatures (,25uC), Y. pestis produces factors that maximize survival and colonization in the flea, such as biofilms [20], while at higher temperatures (.30uC), the bacterium expresses genes required for mammalian infection, including the adhesin Ail, the F1 fimbrial antigen (Caf1), the outer membrane protease Pla, and the Yop-Ysc type III secretion system (T3SS) [2124]. Thus, Y. pestis possesses a variety of virulence factors, including a number of outer membrane-associated factors, which are necessary for interacting with its hosts to ultimately cause disease. Among these, the Pla protease is necessary for the progression of both bubonic and pneumonic plague, but is dispensable during septicemic plague [21,25,26]. Pla is known to cleave a number of mammalian host proteins, including the zymogen plasminogen (plg), the plasmin inhibitor a2-antiplasmin, and the recently identified substrate Fas ligand (FasL), a major inducer of host cell death via apoptosis [2731]. In addition, Pla has also been shown in vitro to act as an adhesin to extracellular matrices by binding laminin as well as promoting the bacterial invasion of HeLa cells [24,32,33]. As Pla is an insoluble outer membrane protein dependent on rough LPS for its protease activity, it is not thought to be secreted by Y. pestis [3436]. However, we have detected active Pla in cellfree culture supernatants, suggesting that this cell-free form of Pla could be contained on OMVs. Here we investigate the ability of Y. pestis to produce native OMVs, characterize the presence and activities of various virulence factors carried on released OMVs, and propose a role for these OMVs during mammalian infection. Outer membrane protein activity in cell-free culture supernatants Our laboratory has detected the activity of the outer membrane protein Pla in cell-free culture supernatants during the exponential growth phase of Y. pestis. To explore this further, 0.2 mm-filtered, cell-free culture supernatants from either wild-type Y. pestis or an isogenic mutant of Y. pestis lacking Pla (Y. pestis Dpla) were grown in the rich media brain-heart infusion (BHI) at 37uC and tested for the ability to convert plg to the active plasmin form, (...truncated)