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Chemokine Degradation by the Group a Streptococcal Serine Proteinase

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Chemokine Degradation by the Group a Streptococcal Serine Proteinase Chemokine degradation by the Group A streptococcal serine proteinase ScpC can be reconstituted in vitro and requires two separate domains Andrea Fritzer, Birgit Noiges, Daniela Schweiger, Angelika Rek, Andreas J. Kungl, Alexander von Gabain, Eszter Nagy, Andreas L. Meinke To cite this version: Andrea Fritzer, Birgit Noiges, Daniela Schweiger, Angelika Rek, Andreas J. Kungl, et al.. Chemokine degradation by the Group A streptococcal serine proteinase ScpC can be reconstituted in vitro and requires two separate domains. Biochemical Journal, Portland Press, 2009, 422 (3), pp.533-542. 10.1042/BJ20090278. hal-00479166 HAL Id: hal-00479166 https://hal.archives-ouvertes.fr/hal-00479166 Submitted on 30 Apr 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Biochemical Journal Immediate Publication. Published on 24 Jun 2009 as manuscript BJ20090278 Chemokine degradation by the Group A streptococcal serine proteinase ScpC can be 2 reconstituted in vitro and requires two separate domains † ‡ †‡ 4 Andrea Fritzer*, Birgit Noiges*, Daniela Schweiger , Angelika Rek , Andreas J. Kungl , § Alexander von Gabain*, Eszter Nagy* and Andreas L. Meinke* 6 8 † *Intercell AG, Campus Vienna Biocenter 3, 1030 Vienna, Austria. Department of 10 Pharmaceutical Sciences, University of Graz, Universitätsplatz 1, 8010 Graz, Austria and ‡ ProtAffin Biotechnologie AG, Reininghausstrasse 13a, 8020 Graz, Austria 12 § corresponding author: Andreas L. Meinke, Intercell AG, Vienna Biocenter 3, A-1030 Vienna, 14 Austria; tel: +43-1-20620-1210; fax: +43-1-20620-18210; e-mail: [email protected] 16 18 Synopsis Streptococcus pyogenes is one of the most common human pathogens and possesses diverse 20 mechanisms to evade the human immune defence. One example of its immune evasion is the degradation of the chemokine IL-8 by ScpC, a serine proteinase that prevents the recruitment of 22 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 24 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 26 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 28 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. 30 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 32 substrates is not restricted to IL-8, since we also detected in vitro protease activity for another CXC chemokine GRO-Į., but not for NAP-2, SDF-1Į, PF-4, I-TAC, IP-10 and MCP-1. The 34 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 36 Spy0416 is a promising vaccine candidate for the prevention of infections by Streptococcus pyogenes. 38 THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20090278 40 Short title: Chemokine degradation by ScpC from S. pyogenes 42 Keywords: Streptococcus pyogenes, Interleukin 8, Spy0416, ScpC, CXC chemokine Accepted Manuscript 1 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited Biochemical Journal Immediate Publication. Published on 24 Jun 2009 as manuscript BJ20090278 Introduction 44 Streptococcus pyogenes belongs to Group A streptococci (GAS) and is mainly an extracellular pathogen capable of causing a wide variety of infections in humans ranging from 46 asymptomatic colonization to life threatening invasive infections [1]. Primary disease manifestations include pharyngitis, impetigo and soft tissue infections, which may lead to 48 invasive diseases if untreated such as sepsis, toxic shock syndrome, rheumatic fever or necrotizing fasciitis [2]. The versatility of this pathogen is explained by complex virulence 50 mechanisms mediated by adhesins such as the M protein and fibronectin binding proteins, as well as powerful invasins e.g. streptolysin O and S [3, 4], and further virulence factors such as 52 streptokinase, hyaluronidase [5] and streptodornase [6]. Additionally, pyrogenic exotoxins which can elicit strong inflammatory cytokine responses leading to endotoxic shock contribute to 54 successful, host-specific survival of S. pyogenes [7]. One of the various strategies the bacterium uses to survive in humans is the antiphagocytic behaviour mediated by binding to fibrinogen via 56 M protein [1, 8-10]. A common molecular mechanism to survive in the human host described for several bacterial pathogens [11, 12] is the secretion of proteolytic enzymes, capable to degrade 58 e.g. immunoglobulins, extracellular matrix and plasma proteins. The virulence factor C5a peptidase e.g. is able to avoid direct contact with phagocytes through destruction of complement- 60 derived chemotaxins [13]. Recently, a novel evasion mechanism, the degradation of the neutrophil recruiting IL-8 62 chemokine was associated with S. pyogenes in patients suffering from necrotizing fasciitis, a severe soft tissue infection associated with high morbidity and mortality rates [14, 15]. The site 64 specific cleavage of IL-8 between Gln59 and Arg60 (converting the 8 kDa IL-8 to a 6 kDa species) was suggested to be responsible for the paucity of neutrophils at the site of infection [14]. It was 66 shown that the cell envelope serine proteinase (ScpC or Spy0416), partially purified from S. pyogenes, was capable of in vitro degradation of human IL-8, GCP-2 and GRO-D, as well as 68 murine keratinocyte-derived chemokine (KC) and murine MIP-2 [16, 17]. These CXC chemokines are responsible for acute neutrophil priming and/or mobilisation and migration in 70 their respective species. In contrast, the CC chemokine RANTES, which is responsible for T-cell and monocyte chemotaxis, was not degraded at all [16]. Furthermore, Spy0416/ScpC was 72 identified as an in vitro expressed and surface exposed protein of GAS by analyses of its surface proteome [18]. The protein comprises 1647 amino acids and appeared to be a highly conserved 74 major component of the cell surface in more than 70% of the analyzed S. pyogenes strains [18]. Although it was not possible to specifically delete the scpC gene, a double mutant in the scpA 76 (encodes the related C5A peptidase) and scpC locus showed drastic reduction of virulence in a murine model of GAS necrotising fasciitis whereas the scpA mutant behaved similar to wild type 78 cells [16]. It was further observed that ScpC promoted resistance to neutrophil killing [19] and contributed to virulence in murine infection models [17, 19, 20]. 80 Here we demonstrate for the first time that the recombinantly expressed form of Spy0416 cleaves IL-8 in a site specific manner that leads to inactivation of this chemokine. Site-directed THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20090278 82 mutagenesis revealed that the amino acid residues in the N-terminal domain, predicted to form the active site of Subtilisin family serine proteases, are indeed essential for enzymatic activity. In 84 addition, we show that the C-terminal domain of Spy0416 for which no function is assigned yet, is also required for proteolytic activity. Besides IL-8 and GCP-2, another CXC chemokine, GRO- 86 Į, is also recognized by recombinant Spy0416 as a specific substrate. Its important function in immune evasion,Accepted the presence in all analyzed S. pyogenesManuscript strains, its high sequence conservation 88 together with its capability to provide protection in animals against challenge with a lethal dose 2 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited Biochemical Journal Immediate Publication. Published on 24 Jun 2009 as manuscript BJ20090278 of S. pyogenes makes (26; unpublished data) Spy0416 an attractive candidate for vaccine 90 development. THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20090278 Accepted Manuscript 3 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited Biochemical Journal Immediate Publication. Published on 24 Jun 2009 as manuscript BJ20090278 Experimental 92 Reagents. The listed reagents were purchased as following: Human chemokines NAP-2, SDF- 94 1D GRO-DMCP-1, IP-10 and I-TAC (PeproTech Inc., UK); recombinant human IL-8 and polyclonal goat anti-human CXCL8/IL-8, CXCL11/I-TAC and IP-10 antibodies (R&D Systems, 96 USA); polyclonal rabbit anti-human IL-8, PF-4, NAP-2 and GRO-Į antibodies (Abcam, UK); goat polyclonal antibodies to SDF-1 and MCP-1 (Santa Cruz Biotechnology, Inc., USA); anti- 98 rabbit IgG Peroxidase conjugated antibody (Sigma, USA); polyclonal rabbit anti-goat IgG-HRP antibody (DakoCytomation, Denmark); monoclonal mouse penta-his antibody (Qiagen, The 100 Netherlands); Ni-SepharoseTM 6 Fast Flow beads (GE Healthcare Biosciences AG, UK); Wide Range Marker (Sigma-Aldrich, USA); PageRuler™ pre-stained protein ladder (Fermentas, 102 Canada); Simply Blue Safe Stain Solution (Invitrogen, USA); Ponceau S stain (Sigma-Aldrich, USA); ECL system (Amersham Pharmacia Biotech, UK). Genomic DNA from S. pyogenes 104 isolates was purified using Wizard® Genomic DNA Purification Kit (Promega, USA).
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