Thromb Haemost 2018; 118(04): 745-757
DOI: 10.1055/s-0038-1637735
Blood Cells, Inflammation and Infection
Schattauer GmbH Stuttgart

Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation

Ulrike Binsker
1   Department of Molecular Genetics and Infection Biology, Interfaculty Institute for Genetics and Functional Genomics, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
,
Raghavendra Palankar
2   Department of Transfusion Medicine, Institute of Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany
,
Jan Wesche
2   Department of Transfusion Medicine, Institute of Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany
,
Thomas P. Kohler
1   Department of Molecular Genetics and Infection Biology, Interfaculty Institute for Genetics and Functional Genomics, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
,
Josephine Prucha
1   Department of Molecular Genetics and Infection Biology, Interfaculty Institute for Genetics and Functional Genomics, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
,
Gerhard Burchhardt
1   Department of Molecular Genetics and Infection Biology, Interfaculty Institute for Genetics and Functional Genomics, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
,
Manfred Rohde
3   Central Facility for Microscopy, ZEIM, Helmholtz Centre for Infection Research, Braunschweig, Germany
,
Frank Schmidt
4   Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, Center for Functional Genomics of Microbes, University Medicine Greifswald, Greifswald, Germany
,
Barbara M. Bröker
5   Department of Immunology, Institute of Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany
,
Uwe Mamat
6   Division of Cellular Microbiology, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany
,
Jan Pané-Farré
7   Department of Microbial Physiology and Molecular Biology, Institute for Microbiology, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
,
Anica Graf
7   Department of Microbial Physiology and Molecular Biology, Institute for Microbiology, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
,
Patrick Ebner
8   Department of Microbial Genetics, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany
,
Andreas Greinacher
2   Department of Transfusion Medicine, Institute of Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany
,
Sven Hammerschmidt
1   Department of Molecular Genetics and Infection Biology, Interfaculty Institute for Genetics and Functional Genomics, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
› Author Affiliations
Funding This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB/TRR34 project C10 to S.H. and A.G., project C4 to B.M.B.). The work was also partially supported by the BMBF/“Unternehmen Region” as part of the ZIK-FunGene.
Further Information

Publication History

05 October 2017

30 January 2018

Publication Date:
19 March 2018 (online)

Abstract

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection.

Supplementary Material

 
  • References

  • 1 Ghoshal K, Bhattacharyya M. Overview of platelet physiology: its hemostatic and nonhemostatic role in disease pathogenesis. Sci World J 2014; 2014: 781857
  • 2 Semple JW, Italiano Jr JE, Freedman J. Platelets and the immune continuum. Nat Rev Immunol 2011; 11 (04) 264-274
  • 3 Cox D, Kerrigan SW, Watson SP. Platelets and the innate immune system: mechanisms of bacterial-induced platelet activation. J Thromb Haemost 2011; 9 (06) 1097-1107
  • 4 Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler Jr VG. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 2015; 28 (03) 603-661
  • 5 Hu L, Umeda A, Kondo S, Amako K. Typing of Staphylococcus aureus colonising human nasal carriers by pulsed-field gel electrophoresis. J Med Microbiol 1995; 42 (02) 127-132
  • 6 Kluytmans J, van Belkum A, Verbrugh H. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev 1997; 10 (03) 505-520
  • 7 Murdoch DR, Corey GR, Hoen B. , et al; International Collaboration on Endocarditis-Prospective Cohort Study (ICE-PCS) Investigators. Clinical presentation, etiology, and outcome of infective endocarditis in the 21st century: the International Collaboration on Endocarditis-Prospective Cohort Study. Arch Intern Med 2009; 169 (05) 463-473
  • 8 Chang FY. Staphylococcus aureus bacteremia and endocarditis. J Microbiol Immunol Infect 2000; 33 (02) 63-68
  • 9 Fowler Jr VG, Miro JM, Hoen B. , et al; ICE Investigators. Staphylococcus aureus endocarditis: a consequence of medical progress. JAMA 2005; 293 (24) 3012-3021
  • 10 Federspiel JJ, Stearns SC, Peppercorn AF, Chu VH, Fowler Jr VG. Increasing US rates of endocarditis with Staphylococcus aureus: 1999-2008. Arch Intern Med 2012; 172 (04) 363-365
  • 11 Siboo IR, Cheung AL, Bayer AS, Sullam PM. Clumping factor A mediates binding of Staphylococcus aureus to human platelets. Infect Immun 2001; 69 (05) 3120-3127
  • 12 Loughman A, Fitzgerald JR, Brennan MP. , et al. Roles for fibrinogen, immunoglobulin and complement in platelet activation promoted by Staphylococcus aureus clumping factor A. Mol Microbiol 2005; 57 (03) 804-818
  • 13 Fitzgerald JR, Loughman A, Keane F. , et al. Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgG binding to the FcgammaRIIa receptor. Mol Microbiol 2006; 59 (01) 212-230
  • 14 Bertling A, Niemann S, Hussain M. , et al. Staphylococcal extracellular adherence protein induces platelet activation by stimulation of thiol isomerases. Arterioscler Thromb Vasc Biol 2012; 32 (08) 1979-1990
  • 15 Eichler P, Budde U, Haas S. , et al. First workshop for detection of heparin-induced antibodies: validation of the heparin-induced platelet-activation test (HIPA) in comparison with a PF4/heparin ELISA. Thromb Haemost 1999; 81 (04) 625-629
  • 16 Biswas R, Voggu L, Simon UK, Hentschel P, Thumm G, Götz F. Activity of the major staphylococcal autolysin Atl. FEMS Microbiol Lett 2006; 259 (02) 260-268
  • 17 De Cuyper IM, Meinders M, van de Vijver E. , et al. A novel flow cytometry-based platelet aggregation assay. Blood 2013; 121 (10) e70-e80
  • 18 Zoll S, Schlag M, Shkumatov AV. , et al. Ligand-binding properties and conformational dynamics of autolysin repeat domains in staphylococcal cell wall recognition. J Bacteriol 2012; 194 (15) 3789-3802
  • 19 Kohler TP, Gisch N, Binsker U. , et al. Repeating structures of the major staphylococcal autolysin are essential for the interaction with human thrombospondin 1 and vitronectin. J Biol Chem 2014; 289 (07) 4070-4082
  • 20 Stentzel S, Sundaramoorthy N, Michalik S. , et al. Specific serum IgG at diagnosis of Staphylococcus aureus bloodstream invasion is correlated with disease progression. J Proteomics 2015; 128: 1-7
  • 21 Schmidt F, Meyer T, Sundaramoorthy N. , et al. Characterization of human and Staphylococcus aureus proteins in respiratory mucosa by in vivo- and immunoproteomics. J Proteomics 2017; 155: 31-39
  • 22 Hussain M, Becker K, von Eiff C, Peters G, Herrmann M. Analogs of Eap protein are conserved and prevalent in clinical Staphylococcus aureus isolates. Clin Diagn Lab Immunol 2001; 8 (06) 1271-1276
  • 23 Albrecht T, Raue S, Rosenstein R, Nieselt K, Götz F. Phylogeny of the staphylococcal major autolysin and its use in genus and species typing. J Bacteriol 2012; 194 (10) 2630-2636
  • 24 Oshida T, Takano M, Sugai M, Suginaka H, Matsushita T. Expression analysis of the autolysin gene (atl) of Staphylococcus aureus. Microbiol Immunol 1998; 42 (09) 655-659
  • 25 Yamada S, Sugai M, Komatsuzawa H. , et al. An autolysin ring associated with cell separation of Staphylococcus aureus. J Bacteriol 1996; 178 (06) 1565-1571
  • 26 Heilmann C, Hussain M, Peters G, Götz F. Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Mol Microbiol 1997; 24 (05) 1013-1024
  • 27 Hirschhausen N, Schlesier T, Schmidt MA, Götz F, Peters G, Heilmann C. A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor. Cell Microbiol 2010; 12 (12) 1746-1764
  • 28 Palma M, Haggar A, Flock JI. Adherence of Staphylococcus aureus is enhanced by an endogenous secreted protein with broad binding activity. J Bacteriol 1999; 181 (09) 2840-2845
  • 29 Hussain M, Haggar A, Peters G. , et al. More than one tandem repeat domain of the extracellular adherence protein of Staphylococcus aureus is required for aggregation, adherence, and host cell invasion but not for leukocyte activation. Infect Immun 2008; 76 (12) 5615-5623
  • 30 Chavakis T, Hussain M, Kanse SM. , et al. Staphylococcus aureus extracellular adherence protein serves as anti-inflammatory factor by inhibiting the recruitment of host leukocytes. Nat Med 2002; 8 (07) 687-693
  • 31 Hussain M, Haggar A, Heilmann C, Peters G, Flock JI, Herrmann M. Insertional inactivation of Eap in Staphylococcus aureus strain Newman confers reduced staphylococcal binding to fibroblasts. Infect Immun 2002; 70 (06) 2933-2940
  • 32 Haggar A, Hussain M, Lönnies H, Herrmann M, Norrby-Teglund A, Flock JI. Extracellular adherence protein from Staphylococcus aureus enhances internalization into eukaryotic cells. Infect Immun 2003; 71 (05) 2310-2317
  • 33 Wang H, von Rohrscheidt J, Roehrbein J. , et al. Extracellular adherence protein of Staphylococcus aureus suppresses disease by inhibiting T-cell recruitment in a mouse model of psoriasis. J Invest Dermatol 2010; 130 (03) 743-754
  • 34 Bur S, Preissner KT, Herrmann M, Bischoff M. The Staphylococcus aureus extracellular adherence protein promotes bacterial internalization by keratinocytes independent of fibronectin-binding proteins. J Invest Dermatol 2013; 133 (08) 2004-2012
  • 35 Woehl JL, Stapels DAC, Garcia BL. , et al. The extracellular adherence protein from Staphylococcus aureus inhibits the classical and lectin pathways of complement by blocking formation of the C3 proconvertase. J Immunol 2014; 193 (12) 6161-6171
  • 36 Joost I, Jacob S, Utermöhlen O. , et al. Antibody response to the extracellular adherence protein (Eap) of Staphylococcus aureus in healthy and infected individuals. FEMS Immunol Med Microbiol 2011; 62 (01) 23-31
  • 37 Komatsuzawa H, Sugai M, Nakashima S. , et al. Subcellular localization of the major autolysin, ATL and its processed proteins in Staphylococcus aureus. Microbiol Immunol 1997; 41 (06) 469-479
  • 38 Baba T, Schneewind O. Targeting of muralytic enzymes to the cell division site of Gram-positive bacteria: repeat domains direct autolysin to the equatorial surface ring of Staphylococcus aureus. EMBO J 1998; 17 (16) 4639-4646
  • 39 Woehl JL, Ramyar KX, Katz BB, Walker JK, Geisbrecht BV. The structural basis for inhibition of the classical and lectin complement pathways by S. aureus extracellular adherence protein. Protein Sci 2017; 26 (08) 1595-1608
  • 40 Prat C, Bestebroer J, de Haas CJ, van Strijp JA, van Kessel KP. A new staphylococcal anti-inflammatory protein that antagonizes the formyl peptide receptor-like 1. J Immunol 2006; 177 (11) 8017-8026
  • 41 de Haas CJ, Veldkamp KE, Peschel A. , et al. Chemotaxis inhibitory protein of Staphylococcus aureus, a bacterial antiinflammatory agent. J Exp Med 2004; 199 (05) 687-695
  • 42 Rooijakkers SH, Ruyken M, van Roon J, van Kessel KP, van Strijp JA, van Wamel WJ. Early expression of SCIN and CHIPS drives instant immune evasion by Staphylococcus aureus. Cell Microbiol 2006; 8 (08) 1282-1293
  • 43 Haas PJ, de Haas CJ, Poppelier MJ. , et al. The structure of the C5a receptor-blocking domain of chemotaxis inhibitory protein of Staphylococcus aureus is related to a group of immune evasive molecules. J Mol Biol 2005; 353 (04) 859-872
  • 44 Stoll H, Dengjel J, Nerz C, Götz F. Staphylococcus aureus deficient in lipidation of prelipoproteins is attenuated in growth and immune activation. Infect Immun 2005; 73 (04) 2411-2423