Erysipelas Caused by Group A Streptococcus Activates the Contact System and Induces the Release of Heparin-Binding Protein Adam Linder, Linda Johansson,

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Erysipelas Caused by Group A Streptococcus Activates the Contact System and Induces the Release of Heparin-Binding Protein Adam Linder, Linda Johansson, Pontus Thulin, Erika Hertzén, Matthias Mörgelin, Bertil Christensson, Lars Björck, Anna Norrby-Teglund, Per Åkesson Journal of Investigative Dermatology Volume 130, Issue 5, Pages 1365-1372 (May 2010) DOI: 10.1038/jid.2009.437 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Proteolytic cleavage of HK in skin biopsies. Proteins were extracted from skin biopsies from the leg with signs of inflammation (a) or of the other noninfected leg (b) and subjected to SDS–PAGE. The gels were transferred to a polyvinylidene difluoride membrane and probed with polyclonal antibodies raised against high-molecular-weight kininogen (HK). Patients are numbered 1–12. Lanes marked “P” contain human plasma stimulated with dextran sulfate, leading to degradation of intact HK. Journal of Investigative Dermatology 2010 130, 1365-1372DOI: (10.1038/jid.2009.437) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Presence of heparin-binding protein (HBP) in skin biopsies. Extracted proteins from tissue taken from infected skin (a) or the contralateral healthy-looking leg (b) of patients 1–12 were analyzed by immunoblotting using a polyclonal antiserum against HBP. Lanes marked “H” contain purified recombinant HBP. Journal of Investigative Dermatology 2010 130, 1365-1372DOI: (10.1038/jid.2009.437) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Host–pathogen responses at the local tissue site of infection. Tissue biopsies from the inflamed leg were obtained from the epicenter, 5cm outside the lesion, and from the healthy leg of patient 11. Biopsies were sectioned, stained for group A streptococci (GAS), streptococcal pyrogenic exotoxin B (SpeB), bradykinin-1 receptor (BK-R1), heparin-binding protein (HBP), and analyzed by in situ imaging as described in Material and Methods. A control staining where the primary antibody was omitted was also performed (ctrl). (a) Representative immunohistochemically stained areas, (b) acquired computerized image analysis (ACIA) values of the whole tissue section. Group A streptococci were also identified by immunofluorescence staining and confocal microscopy analyses (c). Note the distinct positively stained cocci indicated by arrows. Scale bars=30μm (a) and 10μm (c). Journal of Investigative Dermatology 2010 130, 1365-1372DOI: (10.1038/jid.2009.437) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Transmission electron microscopy of thin sections taken from the skin of three patients with erysipelas. (a) Spherical bacteria-like structures are occasionally seen in the dermis. Sections were incubated with a specific antiserum against the Lancefield group A carbohydrate (b) or an antiserum raised against the conserved C-terminal region of the M1 protein (c), followed by detection with gold-labeled secondary antibodies. Colocalization of group-A-specific carbohydrate and M protein with bacterial cell surfaces is visible. (d) Sections were also incubated with gold-labeled fibrinogen. Scale bar=0.5μm. Journal of Investigative Dermatology 2010 130, 1365-1372DOI: (10.1038/jid.2009.437) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions