Thrombin (IIa; 1nM) served as a positive control. (TIF) Characteristics of GAS clinical isolates. (TIFF) Amino acid sequences of PAR reporter constructs. (TIFF) Amino acid sequences encoding mutants of PAR1 reporter construct. (TIFF). studies on endogenously expressed PAR-1 using PAR-1 blocking antibodies further supported our conclusion. Through PAR-1 cleavage SpeB efficiently blunted thrombin-induced induction of the ERK-pathway in endothelial cells and prevented platelets aggregation in response to thrombin. == Conclusions/Significance == Our results identify a novel function of the streptococcal virulence factor SpeB. By cleaving human PAR-1 at the N-terminal amino acid residue leucine44SpeB rendered endothelial cells unresponsive to thrombin BMS-817378 and prevented human platelets from thrombin-induced aggregation. These results suggest that by blunting PAR-1 signaling, SpeB modulates various innate host responses directed against invasive GAS potentially helping the invasive bacteria to escape. This may allow to tailor additional treatments in the future since upon invasion of the blood stream endothelial cells as well as platelets and mononuclear cells respond to PAR-1 agonists aiming to prevent further bacterial dissemination. == Introduction == Group A Streptococcus (GAS) is one of the top ten pathogens causing infection-related deaths world-wide and is responsible for around 0.5 million deaths annually [1]. GAS has evolved a variety of virulence factors facilitating efficient host colonization and invasion [2]. GAS makes use of the hosts clotting network to increase its virulence. Plasminogen activation and recruitment by GAS was for example found to promote the pathogens capability to overcome the hosts barriers and to facilitate blood stream infection [3]. Also the hosts capacity to form fibrin clots is crucial to contain bacterial spread after blood stream invasion [4,5]. On the other hand increased clot formation was shown to boost bacterial dissemination [6]. Recently, studies exploring the bacteria-host interaction from the hosts side showed that a clotting enzyme receptor, the protease activated receptor (PAR)-1, impairs survival in a mousepneumococcalpneumonia model [5] further underlining the interplay between bacteria and the hosts clotting components in the modulation of bacterial virulence. PARs consist of a family of 4 highly related G protein-coupled receptors, abundantly expressed on almost all mammalian cells [7]. PARs allow cells to sense for extracellular enzymatic activity [8] through a unique proteolytic receptor activation mechanism. PAR molecules contain hidden activation ligands within their extracellular N-terminus. Proteolytic removal of N-terminal peptides expose neo-amino N-termini that serve as tethered ligands either activating the same receptor molecule [9] or an adjacent PAR molecule [10], thereby initiating transmembrane signaling. Recently PAR-1 was shown Rabbit polyclonal to HYAL1 to carry several cleavage sites which uncover various signalling-competent tethered ligands causing ligand-specific biological effects [1113]. The impact of PAR-1 activation by mammalian proteases and the resulting effects on systemic inflammation has been extensively studied [1419]. This receptor was found to have important effects on regulating and maintaining the vascular barrier integrity [18], cytokine secretion [20], apoptosis [11,14] and cell proliferation [21]. However studies on how the initiators of systemic inflammation such as bacterial pathogens impact PAR-1 are scarce [22]. So far it was found that the pathogenPorphyromonas gingivaliscausing local infections such as periodonitis promotes platelet activation [23] and thatPseudomonas aeruguinosaactivates PAR-1 and mediates thrombin-like biological effects [24]. However, to our knowledge, major human Gram positive bacterial pathogens responsible for the majority of systemic bacterial infections and consecutive systemic inflammation have not yet been reported to affect PAR-1. Herein we studied whether the human bacterial pathogen GAS responsible for up to 0.66 million yearly systemic infections worldwide [1], affects PAR-1. We found that the GAS secreted cysteine protease streptococcal pyrogenic exotoxin B (SpeB) efficiently cleaved PAR-1. We BMS-817378 identified its specific cleavage site and studied biological downstream effects. We showed that SpeB attenuated extracellular-signal-regulated kinase (ERK) phosphorylation and rendered PAR-1 unresponsive to thrombin and thereby blunted platelet activation. == Results == == Specific cleavage of PAR-1 BMS-817378 by group A streptococcal supernatants == In order to test whether GAS secretes proteases capable of cleaving PARs we relied on a cleavage reporter system we had previously used to characterize human serine proteases [11,25]. In brief all four human PARs encoding mRNAs were cloned.
