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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    • "While SpyCEP is cell wall anchored, it can be shed into the supernatants of bacterial cultures during stationary-phase growth (Chiappini et al., 2012), presumably as a result of protease processing. In addition to these protease activities, SpyCEP also promotes the uptake of GAS into endothelial, but not epithelial, cells (Kaur et al., 2010). Homologous proteases have also been characterized in S. iniae (Zinkernagel et al., 2008) and S. equi (Turner et al., 2009b) and are present in the genomes of several other Streptococcus species (S. dysgalactiae, S. canis, S. didelphis, Streptococcus phocae, S. porcinus, S. pseudoporcinus and Streptococcus thermophilus). "
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    ABSTRACT: Group A Streptococcus (Streptococcus pyogenes; GAS), group B Streptococcus (Streptococcus agalactiae; GBS) and Streptococcus pneumoniae (pneumococcus) are host-adapted bacterial pathogens among the leading infectious causes of human morbidity and mortality. These microbes and related members of the genus Streptococcus produce an array of toxins that act against human cells or tissues, resulting in impaired immune responses and subversion of host physiological processes to benefit the invading microorganism. This toxin repertoire includes hemolysins, proteases, superantigens and other agents that ultimately enhance colonization and survival within the host and promote dissemination of the pathogen. This article is protected by copyright. All rights reserved.
    Preview · Article · Oct 2015 · Cellular Microbiology
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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.
    Full-text · Article · Jul 2014 · Frontiers in Microbiology
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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.
    Full-text · Article · Feb 2013 · Journal of Molecular Medicine
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