The CXC Chemokine-degrading Protease SpyCep of Streptococcus pyogenes Promotes Its Uptake into Endothelial Cells

Department of Microbial Pathogenesis, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2010; 285(36):27798-805. DOI: 10.1074/jbc.M109.098053
Source: PubMed


Streptococcus pyogenes expresses the LPXTG motif-containing cell envelope serine protease SpyCep (also called ScpC, PrtS) that degrades and inactivates the major chemoattractant interleukin 8 (IL-8), thereby impairing host neutrophil recruitment. In this study, we identified a novel function of SpyCep: the ability to mediate uptake into primary human endothelial cells. SpyCep triggered its uptake into endothelial cells but not into human epithelial cells originating from pharynx or lung, indicating an endothelial cell-specific uptake mechanism. SpyCep mediated cellular invasion by an endosomal/lysosomal pathway distinct from the caveolae-mediated invasion pathway of S. pyogenes. Recombinant expression and purification of proteolytically active SpyCep and a series of subfragments allowed functional dissection of the domains responsible for endothelial cell invasion and IL-8 degradation. The N-terminal PR domain was sufficient to mediate endothelial cell invasion, whereas for IL-8-degrading activity, the protease domain and the flanking A domain were required. A polyclonal rabbit serum raised against the recombinant protease efficiently blocked the invasion-mediating activity of SpyCep but not its proteolytic function, further indicating that SpyCep-mediated internalization is independent from its enzymatic activity. SpyCep may thus specifically mediate its own uptake as secreted protein into human endothelial cells.

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    • "In an artificial system based on polystyrene beads coated with recombinant SpyCEP, SpyCEP was found to promote uptake of the beads by HUVECs cells (Kaur et al., 2010). No attachment or internalization was however observed for the epithelial cell lines Hep-2 and A549 and SpyCEP did not mediate invasion when heterologously expressed in Group B streptococci (Kaur et al., 2010). This underlines the fact that an artificial system constituted by beads coated with a single protein can act very differently from live bacteria where many more factors present on the cell surface come into play. "
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    ABSTRACT: SpyCEP-mediated chemokine degradation translates into more efficient spreading and increased severity of invasive Group A Streptococcus (GAS) infections, due to impaired neutrophil recruitment to the site of infection. SpyCEP is markedly up-regulated in invasive as compared to colonizing GAS isolates raising the question whether SpyCEP expression hinders bacterial attachment and thus colonization of the host. To address this question we used a molecular approach involving the use of homologous GAS strains either expressing or not SpyCEP or expressing an enzymatically inactive variant of SpyCEP. We found that expression of enzymatically functional SpyCEP lowered GAS adherence and invasion potential toward various epithelial and endothelial cells. SpyCEP also blunted biofilm formation capacity. Our data indicate that expression of SpyCEP decreases colonization and thus might be detrimental for the spreading of GAS.
    Frontiers in Microbiology 07/2014; 5:339. DOI:10.3389/fmicb.2014.00339 · 3.99 Impact Factor
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    • "We now provide novel evidence that fibrinogen also supports bacterial-endothelial interactions in the presence of the functional GAS fibrinogen receptor M1. Fibrinogen coating GAS facilitated adherence to and invasion of endothelial cells, both factors important for pathogenicity of invasive infections [30, 31]. Fibrinogen at concentrations as low as 10 μg/ml enhanced GAS virulence when the fibrinogen-binding M1 protein was available. "
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    ABSTRACT: Group A Streptococcus (GAS) is a human pathogen causing a wide range of mild to severe and life-threatening diseases. The GAS M1 protein is a major virulence factor promoting GAS invasiveness and resistance to host innate immune clearance. M1 displays an irregular coiled-coil structure, including the B-repeats that bind fibrinogen. Previously, we found that B-repeat stabilisation generates an idealised version of M1 (M1*) characterised by decreased fibrinogen binding in vitro. To extend these findings based on a soluble truncated version of M1, we now studied the importance of the B-repeat coiled-coil irregularities in full length M1 and M1* expressed in live GAS and tested whether the modulation of M1–fibrinogen interactions would open up novel therapeutic approaches. We found that altering either the M1 structure on the GAS cell surface or removing its target host protein fibrinogen blunted GAS virulence. GAS expressing M1* showed an impaired ability to adhere to and to invade human endothelial cells, was more readily killed by whole blood or neutrophils and most importantly was less virulent in a murine necrotising fasciitis model. M1-mediated virulence of wild-type GAS was strictly dependent on the presence and concentration of fibrinogen complementing our finding that M1–fibrinogen interactions are crucial for GAS virulence. Consistently blocking M1–fibrinogen interactions by fragment D reduced GAS virulence in vitro and in vivo. This supports our conclusion that M1–fibrinogen interactions are crucial for GAS virulence and that interference may open up novel complementary treatment options for GAS infections caused by the leading invasive GAS strain M1. Electronic supplementary material The online version of this article (doi:10.1007/s00109-013-1012-6) contains supplementary material, which is available to authorized users.
    Journal of Molecular Medicine 02/2013; 91(7). DOI:10.1007/s00109-013-1012-6 · 5.11 Impact Factor
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    • "The domain architecture of SpyCEP comprises a subtilisin-like protease domain in the N-terminal part of the protein, and a C-terminal region of unknown function followed by a canonical LPXTG cell wall attachment motif. Recombinant expression of SpyCEP fragments showed that the protease domain alone is not sufficient for cleavage of IL-8 in vitro, but that at least the proximal part of the C-terminal needs to be present [12], [13]. We recently showed that the protein exhibits atypical auto processing and that one of the residues in the catalytic triad is located in an N-terminal fragment of the protein, which is released upon autocatalytic hydrolysis. "
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    ABSTRACT: Streptococcus pyogenes is a major human pathogen worldwide, responsible for both local and systemic infections. These bacteria express the subtilisin-like protease SpyCEP which cleaves human IL-8 and related chemokines. We show that localization of SpyCEP is growth-phase and strain dependent. Significant shedding was observed only in a strain naturally overexpressing SpyCEP, and shedding was not dependent on SpyCEP autoproteolytic activity. Surface-bound SpyCEP in two different strains was capable of cleaving IL-8. To investigate SpyCEP action in vivo, we adapted the mouse air pouch model of infection for parallel quantification of bacterial growth, host immune cell recruitment and chemokine levels in situ. In response to infection, the predominant cells recruited were neutrophils, monocytes and eosinophils. Concomitantly, the chemokines KC, LIX, and MIP-2 in situ were drastically increased in mice infected with the SpyCEP knockout strain, and growth of this mutant strain was reduced compared to the wild type. SpyCEP has been described as a potential vaccine candidate against S. pyogenes, and we showed that surface-associated SpyCEP was recognized by specific antibodies. In vitro, such antibodies also counteracted the inhibitory effects of SpyCEP on chemokine mediated PMN recruitment. Thus, α-SpyCEP antibodies may benefit the host both directly by enabling opsonophagocytosis, and indirectly, by neutralizing an important virulence factor. The animal model we employed shows promise for broad application in the study of bacterial pathogenesis.
    PLoS ONE 07/2012; 7(7):e40411. DOI:10.1371/journal.pone.0040411 · 3.23 Impact Factor
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