Lipoproteins in Staphylococcus aureus Mediate Inflammation by TLR2 and Iron-Dependent Growth In Vivo 1 Mathias Schmaler,* Naja J. Jann,* Fabrizia Ferracin,* Lea Z. Landolt,* Lalitha Biswas, † Friedrich Go ¨tz, † and Regine Landmann 2 * Lipoproteins (Lpp) are ligands of TLR2 and signal by the adaptor MyD88. As part of the bacterial cell envelope, Lpp are mainly involved in nutrient acquisition for Staphylococcus aureus. The impact of Lpp on TLR2-MyD88 activation for S. aureus in systemic infection is unknown. S. aureus strain SA113 deficient in the enzyme encoded by the prolipoprotein diacylglyceryl transferase gene (lgt), which attaches the lipid anchor to pro-Lpp, was used to study benefits and costs of Lpp maturation. Lpp in S. aureus induced early and strong cytokines by TLR2-MyD88 signaling in murine peritoneal macrophages. Lpp contributed via TLR2 to pathogenesis of sepsis in C57BL/6 mice with IL-1, chemokine-mediated inflammation, and high bacterial numbers. In the absence of MyD88-mediated inflammation, Lpp allowed bacterial clearing from liver devoid of infiltrating cells, but still conferred a strong growth advantage in mice, which was shown to rely on iron uptake and storage in vitro and in vivo. With iron-restricted bacteria, the Lpp-related growth advantage was evident in infection of MyD88 / , but not of C57BL/6, mice. On the other hand, iron overload of the host restored the growth deficit of lgt in MyD88 / , but not in immunocompetent C57BL/6 mice. These results indicate that iron acquisition is improved by Lpp of S. aureus but is counteracted by inflammation. Thus, lipid anchoring is an evolutionary advantage for S. aureus to retain essential proteins for better survival in infection. The Journal of Immunology, 2009, 182: 7110 –7118. S taphylococcus aureus is a frequent cause of life-threatening sepsis. It expresses multiple virulence factors, which con- tribute to its survival in the host and help its evasion from immune responses. Staphylococcal lipoproteins (Lpp) 3 comprise a large family of membrane-anchored proteins. In the S. aureus ge- nome, 50 genes harbor the type II signal sequence typical for Lpp, 35 of which can be associated with a known or predicted function; many of them are part of ABC transporters and are in- volved in nutrient acquisition (1). Predominant Lpp are SitC (2), which is the binding protein of the staphylococcal iron transporter SitABC; PrsA, the peptidyl-prolyl cis-/trans-isomerase involved in protein folding; and OppA, the oligopeptide permease (1). Besides SitC, several characterized staphylococcal Lpp are involved in iron transport as siderophore binding parts of ABC transporters (FhuD1/FhuD2, SirA, SstD) (2–5) and as binding proteins for heme iron and transferrin iron (IsdE and HtsA) (6, 7). Lpp are synthesized as pre-Lpp with an N-terminal signal se- quence containing a conserved C-terminal lipobox. A diacylglyc- eryl moiety is transferred to the invariant C-terminal cysteine by the lipoprotein diacylglyceryl transferase (Lgt). The signal pep- tide of pro-Lpp is cleaved by the lipoprotein signal peptidase (LspA) (8). While in Gram-negative bacteria Lpp are further modified at the diacylglyceryl-cysteine by N-acyltransferase (Lnt), a homolog of Lnt was not identified in S. aureus, sug- gesting diacylated Lpp. The N-terminal part of an isolated staphylococcal Lpp was characterized as diacylated protein (9), whereas another study found evidence for triacylation of the Lpp SitC by a yet unknown enzyme (10). Lpp purified from several pathogens, including S. aureus, as well as the synthetic lipopeptides Pam 3 CysSerLys 4 (Pam 3 Cys) (11, 12) and Pam 2 CysSerLys 4 (Pam 2 Cys), mirroring the tri- and diacylated Lpp of bacteria, are known to activate TLR2 (10, 13– 18). The three lipid chains of Pam 3 Cys mediate the heterodimer- ization of the TLR1 and TLR2 receptor (12, 19). Diacylated Lpp signal by TLR2/6 dimers, and crystallization modeling predicted binding of lipid chains to TLR2 and stabilization by TLR6 (19). Activation of TLR2 by synthetic lipopeptides leads to induction of cytokines, chemokines, adhesins, and nitrite in macrophages and epithelial cells (11). The complexity of TLR2 in recognition be- comes obvious since staphylococcal lipoteichoic acids (LTA) have been shown to trigger the immune response (20 –22). However, Lpp rather than LTA seem to be the dominant TLR2 stimuli in S. aureus strains (1, 10, 13, 18, 23). Inactivation of Lpp maturation in S. aureus, Mycobacterium tu- berculosis, Listeria monocytogenes, Streptococcus pneumoniae, and Bacillus subtilis resulted in reduced growth under stress con- ditions or in the presence of phagocytes in vitro (1, 23–27). Moreover, S. aureus and group B streptococci lgt gene deletion mutants (lgt) were found to release abundant pre-Lpp possibly by shedding precursor Lpp (1, 28). Lpp maturation is required for virulence of M. tuberculosis and S. pneumoniae in vivo (25, 26, 29). In contrast, lgt mutants in S. aureus and group B streptococci were hypervirulent in vivo and induced less cyto- kines in vitro (1, 23, 28). *Department of Biomedicine, Division Infection Biology, University Hospital Basel, Basel, Switzerland; and † Microbial Genetics, University Tu ¨bingen, Germany Received for publication December 22, 2008. Accepted for publication March 23, 2009. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Swiss National Science Foundation Grants nos. 3100A0-104259/1 and /2 and 3100A0-120617, as well as by Deutsche Forschungs- gemeinschaft Sonderforschungsbereich 766. 2 Address correspondence and reprint requests to Dr. Regine Landmann, Department of Biomedicine, Division Infection Biology, University Hospital Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland. E-mail address: regine.landmann@unibas.ch 3 Abbreviations used in this paper: Lpp, lipoprotein; DIP, dipridyl; lgt, prolipoprotein diacylglyceryl transferase gene; lgt, lgt gene deletion mutant; LTA, lipoteichoic acid; MHA, Mueller-Hinton agar; wt, wild type. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 The Journal of Immunology www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804292