Chemokine degradation by the Group A streptococcal serine proteinase ScpC can be reconstituted in vitro and requires two separate domains

Abstract

Streptococcus pyogenes is one of the most common human pathogens and possesses diverse mechanisms to evade the human immune defence. One example of its immune evasion is the degradation of the chemokine IL (interleukin)-8 by ScpC, a serine proteinase that prevents the recruitment of neutrophils to an infection site. By applying the ANTIGENome technology and using human serum antibodies, we identified Spy0416, annotated as ScpC, as a prominent antigen that induces protective immune responses in animals. We demonstrate here for the first time that the recombinant form of Spy0416 is capable of IL-8 degradation in vitro in a concentration- and time-dependent manner. Mutations in the conserved amino acid residues of the catalytic triad of Spy0416 completely abolished in vitro activity. However, the isolated predicted proteinase domain does not exhibit IL-8-degrading activity, but is dependent on the presence of the C-terminal region of Spy0416. Binding to IL-8 is mainly mediated by the catalytic domain. However, the C-terminal region modulates substrate binding, indicating that the proteolytic activity is amenable to regulation via the non-catalytic regions. The specificity for human substrates is not restricted to IL-8, since we also detected in vitro protease activity for another CXC chemokine GRO-alpha (growth-related oncogene alpha), but not for NAP-2 (neutrophil-activating protein 2), SDF (stromal-cell-derived factor)-1alpha, PF-4 (platelet factor 4), I-TAC (interferon-gamma-inducible T-cell alpha-chemoattractant), IP-10 (interferon-gamma-inducible protein 10) and MCP-1 (monocyte chemoattractant protein 1). The degradation of two human CXC chemokines in vitro, the high sequence conservation, the immunogenicity of the protein in humans and the shown protection in animal studies suggest that Spy0416 is a promising vaccine candidate for the prevention of infections by S. pyogenes.

Novel Conserved Group A Streptococcal Proteins Identified by the Antigenome Technology as Vaccine Candidates for a Non-M Protein-Based Vaccine

“…In addition, Spy0416 was shown to mediate cleavage of IL-8, thereby impairing clearance from infected tissues and promoting resistance to neutrophil killing (30,66). We provided further evidence that this activity was also exhibited by the recombinant form of the Spy0416 protein and required the concerted actions of the enzymatic and ancillary domains of the large ScpC protein (21). These studies further strengthen the selection of Spy0416 as a candidate for a GAS vaccine.…”

Streptococcal M1 Protein Triggers Farnesyltransferase-Dependent Formation of CXC Chemokines in Alveolar Macrophages and Neutrophil Infiltration of the Lungs

“…Neutrophils are particularly responsive to CXC chemokines, comprising CXCL1 and CXCL2, which are murine homologues of human interleukin-8 (42). Convincing lines of evidence have demonstrated clear-cut formation of CXC chemokines in streptococcal infections (8,15), and we have recently observed that M1 protein triggers significant pulmonary formation of CXCL1 and CXCL2 (40). Thus, we next asked whether farnesyltransferase might regulate M1 protein-induced formation of CXC chemokines in the lung.…”

SpyA, a C3-Like ADP-Ribosyltransferase, Contributes to Virulence in a Mouse Subcutaneous Model of Streptococcus pyogenes Infection

“…Reduction of chemokine levels appears to be an important pathogenic mechanism for invasive GAS. The bacterium encodes a protease, SpyCEP (PrtS, ScpC), capable of cleaving a number of human CXC chemokines in addition to murine CXCL1 and CXCL2; isogenic mutants lacking SpyCEP are less virulent than their wildtype counterparts (16,18,27,33). Continued work is needed to ascertain the specific role, if any, that SpyA-mediated avoidance of uptake plays in epithelial cell chemokine production.…”