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Inactivates Neutrophil Proteins S100A8/A9 Bacterial Surface Bacterial Surface Protein L Binds and Inactivates Neutrophil Proteins S100A8/A9 Bo Åkerström and Lars Björck This information is current as J Immunol 2009; 183:4583-4592; Prepublished online 14 of September 28, 2021. September 2009; doi: 10.4049/jimmunol.0901487 http://www.jimmunol.org/content/183/7/4583 Downloaded from References This article cites 57 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/183/7/4583.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 28, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Bacterial Surface Protein L Binds and Inactivates Neutrophil Proteins S100A8/A91 Bo Åkerstro¨m2 and Lars Bjo¨rck2 Finegoldia magna is an anaerobic bacterial species that is part of the normal human flora on all nonsterile body surfaces, but it is also a significant opportunistic pathogen causing a wide range of infections. Some isolates of F. magna that are more frequently associated with clinical infection express protein L, a surface protein containing multiple homologous domains (B1-B5) that bind Igs through interactions with Ig L chains. The present study shows that the N-terminal A domain of protein L binds S100A8/A9, antibacterial proteins present in large amounts in the cytoplasm of neutrophils, but also extracellularly in tissues during inflammation. As a result, protein L-expressing F. magna are protected against killing by S100A8/A9. Igs and S100A8/A9 were found to interact independently with protein L, demonstrating that this bacterial surface protein is capable of manipulating both adaptive and innate immune defense mechanisms. The Journal of Immunology, 2009, 183: Downloaded from 4583–4592. any bacterial species express Ig-binding surface pro- in the release of proinflammatory mediators (14, 15). These teins, among which staphylococcal protein A (1) and different observations indicate that the presence of protein L at M streptococcal protein G (2, 3) are the most well the surface of F. magna enhances the potential pathogenicity of known. These proteins are widely used as biomedical tools to the bacteria. http://www.jimmunol.org/ detect and bind Abs, and they interact with the H chains of IgG. Members of the human S100 protein family are Ca2ϩ-binding Apart from protein L, an Ig L chain-binding molecule (4), all proteins containing helix-loop-helix motifs, so-called EF hands bacterial proteins to date described that bind Ig in a nonimmune (16). The expression of the S100 proteins is usually regulated fashion interact with Ig H chains. Protein L is expressed by by environmental and developmental factors, and is cell and ϳ10% of clinical isolates of Finegoldia magna (5), an anaer- tissue specific. Two members, S100A8 and S100A9, are major obic Gram-positive bacterium that is part of the normal flora in cytosolic constituents (ϳ40% of the total protein content) in the skin, the mouth, the upper respiratory tract, the gastrointes- neutrophils and monocytes. The apparent molecular masses on tinal tract, and the female genito-urinary tract. F. magna is the SDS-PAGE are 8 and 14 kDa, respectively, and they are also most significant opportunistic pathogen of the anaerobic normal called myeloid-related proteins 8 and 14, migration inhibitory by guest on September 28, 2021 bacterial flora, causing soft tissue abscesses, bone/joint infec- factor-related proteins 8 and 14, or calgranulin a/b (17, 18). tions, wound infections, and vaginosis (6, 7). Protein L-express- Their expression is up-regulated in tissue macrophages, kera- ing F. magna strains are frequently isolated from patients with tinocytes, and epithelial cells in inflammation and cancer (19, vaginosis (5), and when expressed at the surface of Streptococ- 20). S100A8 and S100A9 are secreted from activated phago- cus gordonii, protein L promotes the adhesion of these bacteria cytes by a novel tubulin-dependent mechanism (21), and they to the vaginal mucosa of mice (8). Protein L preferentially binds are found in extracellular fluid and plasma during inflammation Ig L chains of the ␬ type through interactions with the variable (20). The two proteins form heteromeric complexes in vivo, and domain, but without interfering with the Ag binding site (9– at millimolar Ca levels the heterotetramer, (S100A8/A9) ,is 12). The molecule contains multiple homologous Ig binding 2 predominating (22, 23). The tetramer was also named calpro- domains, called B repeats (13), and it activates human basophils tectin due to its ability to inhibit growth of various bacterial and and mast cells by cross-linking surface-associated IgE, resulting fungal species (24). This bacteriostatic property has been as- cribed to the Zn-binding capacity of the tetramer, and it has been suggested that the effect is a result of depletion of bacterial Department of Clinical Sciences, Division of Infection Medicine, Lund University, nutrient Zn ions (25) or altered structure of the protein complex Lund, Sweden (26). The observation that the S100A8/A9 complex adheres to Received for publication May 11, 2009. Accepted for publication July 21, 2009. amastigotes in skin lesions of Leishmania major-infected mice The costs of publication of this article were defrayed in part by the payment of page (27) indicates that S100A8/A9 could also have a function in the charges. This article must therefore be hereby marked advertisement in accordance defense against this parasite. with 18 U.S.C. Section 1734 solely to indicate this fact. The interaction between the B repeats of protein L and Ig L 1 This work was supported by the Swedish Research Council (Projects 7144 and 7480), Swedish Government Funds for Clinical Research (ALF), Swedish Society for chains is well documented, but the properties of the N-terminal A Medical Research, Royal Physiographic Society (Lund), Foundations of Greta and domain (Fig. 3A shows a schematic depiction of protein L) are ¨ Johan Kock, Torsten and Ragnar So¨derberg, Alfred Osterlund, the Blood and Defence unknown. The starting point for the present investigation was the Network, Lund University, the Crafoord Foundation, the Thelma Zoe´ga Foundation, and Hansa Medical AB. finding that a protein L construct containing the A domain bound 2 Address correspondence and reprint requests to Dr. Bo Åkerstro¨m or Dr. Lars S100A8/A9 with high affinity and specificity, whereas the Ig-bind- Bjo¨rck, Lund University, BMC, B14, Solvegatan 19, SE-22184, Lund, Sweden. ing B repeats did not. A characterization of the interaction between E-mail addresses: [email protected] and [email protected] protein L and S100A8/A9 and its consequences for F. magna bi- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 ology is the theme of this work. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901487 4584 PROTEIN L BINDS AND INACTIVATES S100 A8/A9 Materials and Methods some cases, binding experiments were also performed in TH broth con- Bacteria taining 0.5% Tween 80 (pH 5.5). F. magna (strains 312, 505, 564, and 644) and group G streptococcal Bacterial growth assay strains (G42, G43, G46, and G148) are clinical isolates from the De- F. magna strains 312 and 505 were grown under strict anaerobic conditions partment of Clinical Microbiology, Lund University Hospital. Strepto- in TH broth (pH 7.5) containing 0.5% Tween 80, to A620 0.5–0.7. A total coccus pneumoniae strains D39 and PR218 are from G. Pozzi, Univer- of 4 ␮l of these bacterial suspensions was added to 200 ␮l of TH (pH 5.5 sity of Siena, and the Streptococcus pyogenes strains (AP1, AP4, AP6, or 7.5) containing 0.5% Tween 80, in UV-transparent cuvettes. At the same AP12, and AP49) are from Institute of Hygiene and Epidemiology (Prague, time, various amounts of S100A8/A9 in 1–2 ␮l of PBS, or 1–2 ␮lofPBS Czech Republic). The two Staphylococcus aureus strains Cowan I and alone as a control, were added to the cuvettes. These manipulations were Wood 46 are from T. Foster, Trinity College (Dublin, Ireland). All strains 3 done in an anaerobic workstation (Electrotek). A620 was determined after were grown in Todd-Hewitt (TH) broth (Difco) at 37°C. In the case of F. 24 h of incubation. In some experiments, the strains were grown at pH 5.5 magna strains, TH contained 0.5% Tween 80, and these isolates were to A620 0.3–0.5 in the UV-transparent cuvettes, followed by the addition of grown under strict anaerobic conditions. 5 ␮g of S100A8/A9. Growth curves were obtained by cultivating the bac- teria under strict anaerobic conditions and measuring A at different time Proteins, Abs, sequencing, and radiolabeling 620 points. Protein L (B1-B4) was expressed and purified, as described (13). Factor Xa (FXa) was purchased from ICN Pharmaceuticals. Human IgG was pur- Construction and cloning of a protein L (A-C2) vector chased from Sigma-Aldrich. Protein PAB was isolated from F. magna A DNA fragment coding for an N-terminal FXa-site (isoleucine-glutamic (strain ALB8) culture medium, as described (28).
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