Role of Lipoteichoic Acid in the Phagocyte Response to Group B Streptococcus Philipp Henneke, Siegfried Morath, Satoshi Uematsu, Stefan Weichert, Markus Pfitzenmaier, Osamu Takeuchi, Andrea This information is current as Müller, Claire Poyart, Shizuo Akira, Reinhard Berner, of September 28, 2021. Giuseppe Teti, Armin Geyer, Thomas Hartung, Patrick Trieu-Cuot, Dennis L. Kasper and Douglas T. Golenbock J Immunol 2005; 174:6449-6455; ; doi: 10.4049/jimmunol.174.10.6449 Downloaded from http://www.jimmunol.org/content/174/10/6449 References This article cites 48 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/174/10/6449.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Role of Lipoteichoic Acid in the Phagocyte Response to Group B Streptococcus1 Philipp Henneke,3* Siegfried Morath,2† Satoshi Uematsu,2§ Stefan Weichert,* Markus Pfitzenmaier,‡ Osamu Takeuchi,§ Andrea Mu¨ller,* Claire Poyart,¶ Shizuo Akira,§ Reinhard Berner,* Giuseppe Teti,# Armin Geyer,‡ Thomas Hartung,† Patrick Trieu-Cuot,ʈ Dennis L. Kasper,** and Douglas T. Golenbock†† Group B Streptococcus (GBS) cell walls potently activate phagocytes by a largely TLR2-independent mechanism. In contrast, the cell wall component lipoteichoic acid (LTA) from diverse Gram-positive bacterial species has been shown to engage TLR2. In this study we examined the role of LTA from GBS in phagocyte activation and the requirements for TLR-LTA interaction. Using cells from knockout mice and genetic complementation in epithelial cells we found that highly pure LTA from both GBS and Staph- Downloaded from ylococcus aureus interact with TLR2 and TLR6, but not TLR1, in contrast to previous reports. Furthermore, NF-␬B activation by LTA required the integrity of two putative PI3K binding domains within TLR2 and was inhibited by wortmannin, indicating an essential role for PI3K in cellular activation by LTA. However, LTA from GBS proved to be a relatively weak stimulus of phagocytes containing ϳ20% of the activity observed with LTA from Staphylococcus aureus. Structural analysis by nuclear magnetic resonance spectrometry revealed important differences between LTA from GBS and S. aureus, specifically differences in glycosyl linkage, in the glycolipid anchor and a lack of N-acetylglucosamine substituents of the glycerophosphate backbone. http://www.jimmunol.org/ Furthermore, GBS expressing LTA devoid of D-alanine residues, that are essential within immune activation by LTA, exhibited similar inflammatory potency as GBS with alanylated LTA. In conclusion, LTA from GBS is a TLR2/TLR6 ligand that might contribute to secreted GBS activity, but does not contribute significantly to GBS cell wall mediated macrophage activation. The Journal of Immunology, 2005, 174: 6449–6455. roup B Streptococcus (GBS)4 is a common commensal frequent event (2). Early recognition of GBS structures by phago- of the human genital and intestinal tract. Yet GBS is a cyte receptors must ensure immediate and nonphlogistic removal potent inflammatory stimulus once it becomes invasive. of GBS from blood and tissue to prevent the host from succumbing G by guest on September 28, 2021 GBS causes fulminant sepsis in newborn infants and constitutes to bacterial spread and subsequent generalized inflammation. We the third most common cause of bacterial meningitis in countries have previously shown that the evolutionary conserved TLR con- that have implemented immunization with Haemophilus influenzae stitute a highly sensitive receptor system for GBS that determine type b conjugate vaccines (1). the cause of invasive GBS disease in vivo (3–5). However, Because 15% of healthy human beings are fecal carriers of GBS, whereas LPS from Gram-negative bacteria carries most activities the invasion of small numbers of GBS can be assumed to be a of the whole bacteria, the identity of toxins from GBS that mediate the inflammatory response to infection, as with many other Gram- *Zentrum fu¨r Kinderheilkunde und Jugendmedizin, Albert-Ludwigs Universita¨t positive bacteria, is still unclear. Freiburg, Freiburg, †Abteilung fu¨r Biochemische Pharmakologie, Universita¨t Kon- The cell wall of Gram-positive bacteria constitutes a macromo- ‡ stanz, Konstanz, Fachbereich Chemie, Philipps-Universita¨t Marburg, Marburg, Ger- lecular assembly of surface proteins, cross-linked peptidoglycan many; §Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; ¶Laboratoire de Bacte´riologie, Faculte´deMe´decine Cochin, Paris, ʈUnite´de Biologie (PGN), polyanionic teichoic acid, and lipoteichoic acid (LTA) (6). des Pathoge`nes a`Gram-positif, Institut Pasteur, Paris France; #Dipartimento di Pa- Because PGN and LTA, teichoic acid or similar anionic molecules tologia e Microbiologia Sperimentale, Universita`di Messina, Messina, Italy; **Chan- ning Laboratory, Brigham and Women’s Hospital, and Departments of Medicine, are shared by virtually all Gram-positive bacteria, these molecules Microbiology and Molecular Genetics, Boston, MA 02115; ††Department of Medi- are candidates as conserved toxins or “pathogen-associated mo- cine, University of Massachusetts Medical School, Worcester, MA 01605 lecular patterns” that are widely believed to be recognized by Received for publication November 10, 2004. Accepted for publication March TLRs (7). Indeed, LTA and PGN from Staphylococcus aureus 2, 2005. have been demonstrated to interact with TLR2 and the TLR core- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance ceptor CD14 (8, 9). LTA is particularly interesting as a Gram- with 18 U.S.C. Section 1734 solely to indicate this fact. positive “endotoxin” because it resembles LPS in some respects. 1 This work was supported in part by Deutsche Forschungsgemeinschaft He 3127/2-1 LTA and LPS are both anchored via their glycolipids to the mem- (to P.H.), by the National Institutes of Health Grant AI52455 and GM54060 (to D.T.G.), and by the European Union HOSPATH QLRT-1999-30336 (to G.T.). brane and carry a polysaccharide chain extending into the PGN layer of the cell wall. Whereas glycolipids like LTA are widely 2 S.M. and S.U. contributed equally to this work. recognized TLR ligands, PGN preparations, which are a hetero- 3 Address correspondence and reprint requests to Dr. Philipp Henneke, Zentrum fu¨r Kinderheilkunde und Jugendmedizin, Albert-Ludwigs Universita¨t Freiburg, Mathilden- geneous mixture of repeating disaccharide (N-acetylmuramic acid- strasse 1, 79106 Freiburg, Germany. E-mail address: [email protected] (b1–4)-N-acetylglucosamine) containing cell wall fragments, have 4 Abbreviations used in this paper: GBS, group B streptococcus; LTA, lipoteichoic no known TLR2 binding domain and have recently been ques- acid; HEK, human epithelial kidney; PGN, peptidoglycan; NMR, nuclear magnetic resonance; MALP-2, macrophage-activating lipopeptide of 2 kDa; MDP, muramyl tioned as a TLR2 ligand (11). Within the cell wall network LTA is dipeptides. thought to provide elasticity, porosity and tensile strength of the Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 6450 RECOGNITION OF LTA bacterial envelope. However, its contribution to the interaction be- plates at a density of 2 ϫ 104 cells/well. The following day, cells were tween bacteria and cells of the mammalian immune system within transiently transfected with luciferase reporter genes using Fugene (Roche) ␬ invasive infections remains less well understood. As an example, per the manufacturer’s recommendations. To assess NF- B activation, a reporter gene consisting of an E-selectin promoter driving a firefly lucif- we previously demonstrated that cell wall preparations from GBS erase gene was used. In individual experiments, cells were cotransfected activate immune cells in the absence of both TLR2 and CD14. with a constitutively active Renilla luciferase reporter gene (Promega) to These preparations were likely to be rich in LTA, thus raising normalize between conditions for transfection efficacy. The following day, questions about the cognate receptor of LTA from GBS and the the cells were stimulated as indicated. When indicated, HEK293 cells were transfected with human TLR2FLAG, TLR2-A616FLAG, TLR2-A761FLAG, contribution of LTA to phagocyte activation by GBS (3, 4, 12). TLR2-A616/761FLAG, or with empty vector. Expression vectors for TLR2- To answer the question about the significance of PGN and LTA A616FLAG, TLR2-A761FLAG, and TLR2-A616/761FLAG were kind gifts of in GBS infection, we defined several issues: 1) whether LTA from U. Knaus