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

Simran Jeet Kaur,Andreas Nerlich,Simone Bergmann,Manfred Rohde,Marcus Fulde,Dorothea Zähner,Emanuel Hanski,Annelies Zinkernagel,Victor Nizet,Gursharan S Chhatwal,Susanne R Talay

doi:10.1074/jbc.m109.098053

Abstract

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.

Highlights

Streptococcus pyogenes is an important human pathogen able to cause infections ranging from mild pharyngitis to severe invasive disease
A recent study focused on the role of SpyCep in the globally disseminated M1T1 S. pyogenes clone that has emerged as the leading cause of invasive infections during recent epidemiology [5]
Western blotting for direct detection of the fusion protein recognized a band with the anticipated molecular mass of ϳ150 kDa in the cell lysate of E. coli transformants (Fig. 1A), indicating expression of mature full-length recombinant mature full-length SpyCep (rSpyCep). rSpyCep was purified from the bacterial cell lysate under native conditions by using immobilized NiNTA affinity chromatography (Fig. 1B)

Summary

Introduction

Streptococcus pyogenes is an important human pathogen able to cause infections ranging from mild pharyngitis to severe invasive disease. 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.

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Conclusion