Immune responses induced by spirochetal outer membrane lipoproteins and glycolipids

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Immunobiology 213 (2008) 329–340 www.elsevier.de/imbio

REVIEW

Immune responses induced by spirochetal outer membrane lipoproteins and glycolipids Nicolas W.J. Schro¨dera, Jana Eckertb, Gunthard Stu¨bsb, Ralf R. Schumannb, a

Department for Dermatology and Allergology, Charite´-Universita¨tsmedizin Berlin, Charite´platz 1, D-10117 Berlin, Germany Institut fu¨r Mikrobiologie und Hygiene, Charite´-Universita¨tsmedizin Berlin, Dorotheenstr. 96, D-10117 Berlin, Germany

b

Received 3 August 2007; received in revised form 18 October 2007; accepted 1 November 2007

Abstract The class of Spirochetes comprises a wide array of clinically important pathogens, including Treponema pallidum causing syphilis as well as Borrelia burgdorferi, the agent of Lyme disease (LD). Diseases caused by spirochetes are characterized by specific sequelae of host reactions, and also by characteristic antibody response patterns. Over the last decades, research on the interaction of spirochetes with the host’s immune system had a strong emphasis on outer membrane lipoproteins. In fact, these structures have been convincingly shown to activate immune cells via CD14 and Toll-like receptor (TLR)-2, and recent data also indicate an interaction with lipopolysaccharide (LPS)-binding protein (LBP). In particular, the interaction of B. burgdorferi with TLR-2 could not only be demonstrated in mice, but was also supported by data showing that genetic variants of TLR-2 in humans influenced the clinical course of LD. However, there is increasing evidence that next to lipoproteins, glycolipids may also play an important role in responses of the immune system towards spirochetes. Diacylglycerol-containing glycolipids exhibiting similarities with lipoteichoic acid (LTA) of Gram-positive bacteria have been demonstrated in various Treponema species, whereas LPS-like glycolipids have been shown to be present in Leptospira. Treponema glycolipids, comparably to lipoproteins and LTA, interact with LBP, CD14 and TLRs. In contrast, complex glycolipids of high molecular weight could not be demonstrated in Borrelia, whereas these bacteria exhibit a number of unique low molecular weight glycolipids. Some of these glycolipids cause strong immediate immune responses, while others appear to be potent antigens for induction of an adaptive immune response. This review summarizes data obtained so far on amphiphilic and hydrophobic molecules from spirochetes regarding structure and influence on innate as well as adaptive immune responses. r 2007 Elsevier GmbH. All rights reserved. Keywords: Innate immunity; Toll-like receptors; Spirochetes; Lipoproteins; Glycolipids

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 Spirochetes – unique bacteria causing complex diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 The innate immune system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 Abbreviations: ACGal, acylated cholesteryl-galactoside; LBP, lipopolysaccharide binding protein; LD, Lyme disease; LP, lipopeptide; LPS, lipopolysaccharide; LTA, lipoteichoic acid; LRR, Leucin-rich repeat; MGalD, a-monogalactosyl-diacylglycerol; PRR, pattern recognition receptor; NF-kB, nuclear factor kB; TLR, Toll-like receptor. Corresponding author. Tel.: +49 30 450 524141; fax: +49 30 450 524941. E-mail address: [email protected] (R.R. Schumann). 0171-2985/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2007.11.003

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Spirochetal outer membrane proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction of spirochetal lipoproteins with TLRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LBP as binding partner for Osps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence for an influence of TLR-mediated recognition of Borrelia on course of Lyme disease . . . . . . . . . . . . . Glycolipids in Treponema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction with the innate immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LPS in Leptospira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glycolipids in Borrelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Low molecular weight glycolipids in Borrelia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction of MGalD with the innate immune system (NKT cells) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ACGal as a candidate for vaccination approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction Spirochetes – unique bacteria causing complex diseases Spirochetes are small spiral-shaped bacteria with a unique cell wall composition (Johnson, 1977). They include the genera Treponema, Leptospira and Borrelia, which all contain species pathogenic for the human host (Paster and Dewhirst, 2000). Treponema pallidum is the cause of syphilis, and other, i.e. oral Treponema have been shown to be involved in periodontitis and potentially endocarditis (Golden et al., 2003; Moter et al., 1998; Riviere et al., 1991). Leptospires are the cause of Weil’s disease, a rare systemic syndrome consisting of flu-like symptoms that may also lead to meningitis, liver damage and renal failure (Palaniappan et al., 2007). Of the 37 known Borrelia species, 12 can cause Lyme disease (LD) transmitted by ticks (Hengge et al., 2003). Others, such as B. recurrentis or B. hermsil are the cause of relapsing fever. A simple overview of the family of spirochetes is given in Fig. 1. Most of the diseases caused by spirochetes are characterized by different phases including late stage complications, such as neurologic symptoms or arthritis occurring as late stages of LD (Stanek and Strle, 2003). Diagnosis is often not easy and has to rely on serologic parameters due to problems in culturing spirochetes directly (Wilske et al., 2007). Insight in the pathogenesis of diseases has been gained lately by analysis of cellular components causing innate or acquired immune responses of the host. For spirochetes the cell wall until recently has not been analyzed completely, which also has been caused by problems in culturing these bacteria (Yotis et al., 1991). It is known that they contain two membranes organized in a similar fashion as compared to Gram-negative bacteria, thus several reports postulated the presence of lipopolysaccharide (LPS), the typical constituent of the Gram-negative cell wall,

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although formal proof was lacking (Dahle et al., 1996; Habicht et al., 1986; Walker et al., 1999). Recently, however, by deciphering the genomes, it became clear that LPS synthesis genes are lacking, and the presence of LPS was ruled out for Treponema and Borrelia (Fraser et al., 1997, 1998; Norris and Weinstock, 2000; Seshadri et al., 2004; Takayama et al., 1987). Leptospira, in contrast contain an LPS with a unique chemical composition and a specific immune activity (Nahori et al., 2005; Que-Gewirth et al., 2004; Werts et al., 2001). Spirochetes furthermore contain a unique internal flagellum causing the spiral shape and the motility of spirochetes, located between the two membranes of these bacteria (Fig. 2). The composition of the spirochetal cell wall, and the biological activity of the outer membrane proteins will be described in more detail in the following sections.

The innate immune system Microorganisms contain structures that are recognized by the innate immune system of the host initiating immediate defense responses and also the activation of the acquired immune system (Akira et al., 2006). Over the last 10 years, a large number of host molecules have been identified with the ability to recognize bacterial motifs and to initiate inflammatory responses directly, among them the family of Toll-like receptors (TLRs) (Beutler et al., 2006). They contain a Leucin-rich repeat (LRR) domain responsible for ligand binding and due to the ability to recognize microbial ‘‘patterns’’ are referred to as ‘‘pattern recognition receptors (PRRs)’’, a term shared with other receptors such as the scavenger receptor or the mannose receptor (Schro¨der and Schumann, 2005a). TLRs furthermore contain a signal transduction domain similar to the one found in the interleukin-1 receptor. This domain was termed toll/ interleukin-1 receptor (TIR) domain and activation leads to the recruitment of the signal transducer

ARTICLE IN PRESS N.W.J. Schro¨der et al. / Immunobiology 213 (2008) 329–340 Superkingdom

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B. burgdorferi Lyme Disease B. recurrentis B. hermsii

S. hyodysenteriae

L. interrogans Weil's Disease

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T. socranskii T. medium T. maltophilum Periodontitis T. brennaborense Digital Dermatitis

Fig. 1. Taxonomy of Spirochetes. The figure shows a simplified version of taxonomy of spirochetes focusing on the clinically important genera. Bacteria specifically mentioned in the text are boxed; the diseases caused by them or associated with their presence are shown in red.

Lipopolysaccharide (LPS) outer membrane peptidoglycan cytoplasmic membrane Gram-negative Bacteria

glycolipids (LPS*) lipoproteins outer membrane flagellum periplasmic space peptidoglycan cytoplasmic membrane Spirochetes

Fig. 2. Membrane architecture of Gram-negative bacteria in comparison to spirochetes. Gram-negative bacteria possess two outer membranes and a small layer of peptidoglycan. The outer membrane is formed by LPS. Spirochetes also exhibit two membranes, but the outer membrane is not attached to the peptidoglycan layer, and the periplasmic space contains the flagellum. The outer membrane contains several lipoproteins, and glycolipids, in particular LPS, have been repeatedly described to be present as well. Note that LPS has been ruled out for Borrelia and Treponema, but has been shown in Leptospira.

MyD88 that initiates a cascade of events finally leading to translocation of NF-kB into the nucleus (O’Neill and Bowie, 2007).

On the surface of most host cells, TLR2 with its heterodimerization partners TLR1, -6, and potentially -10 is expressed, as well as TLR4, and -5. A large

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number of bacterial, protozoan and viral ligands for the TLR2 complex has been reported, with lipoproteins being the most potent ligands of TLR2 (Kirschning and Schumann, 2002). In light of this, the activation of TLR2 by ligands other than lipoproteins or lipopeptides (LPs) may be at least in part caused by lipoprotein contamination (Ogawa et al., 2007). TLR4 recognizes mainly bacterial LPS, whereas TLR5 is the receptor of bacterial flagellin. TLRs 3, and -7 through 9 are located in endosomes and recognize nucleic acids of viral or microbial origin. Lipoprotein and LP recognition by TLR2 apparently depends on the composition of the heterodimeric TLR2 receptor complex with TLR2/1 heterodimers recognizing triacylated LPs, and the TLR2/6 complex recognizing diacylated LPs (BuwittBeckmann et al., 2005, 2006; Manukyan et al., 2005). Furthermore, in the case of LPs, the amino acid sequence has a strong impact on interaction with TLR2/1 or TLR2/6 heterodimers (Buwitt-Beckmann et al., 2005; Omueti et al., 2005). Besides cellular recognition, humoral innate immune defense systems exist including the complement system and a number of soluble recognition proteins. One protein that will be described in more detail below is the LPS-binding protein (LBP) first described as binding partner for bacterial LPS (Schumann et al., 1990; Tobias et al., 1986). This acute-phase serum protein lately has also been found to recognize Gram-positive bacteria and LPs of different bacterial origin and thus may be viewed as soluble PRR (Schro¨der et al., 2004; Weber et al., 2003).

Spirochetal outer membrane proteins Interaction of spirochetal lipoproteins with TLRs Bacterial lipoproteins early have been shown to be potent activators of the innate immune system. Spirochetes contain specific lipoproteins that also elicit a strong humoral response, which has been instrumental, e.g. for the diagnosis of LD. A group of spirochetal proteins has been termed ‘‘outer surface proteins (Osps)’’ and Osps of Borrelia burgdorferi have been described early to induce cytokines (Ma and Weis, 1993; Radolf et al., 1991; Wooten et al., 1996). Furthermore, immunostimulatory lipoproteins termed p17 and p47 were described in the outer membrane of T. pallidum. Lipoproteins have also been described in the oral spirochete Treponema denticola (Tde) (Sela et al., 1997; Yotis et al., 1991). Lipoproteins from Borrelia spp. as well as synthetic LPs bearing the Pam3Cys moiety were found to interact with TLR2, and not TLR4 as they were shown to be able to stimulate cells isolated from C3H/HeJ mice lacking the TLR4 receptor (Hirschfeld et al., 1999; Lien

et al., 1999; Schwandner et al., 1999). In order to interact with its ligands, TLR2 forms heterodimers either with TLR1 or -6. TLR2/1 heterodimers recognize triacylated lipoproteins and LPs (Alexopoulou et al., 2002; Takeuchi et al., 2002) as has been confirmed recently by co-crystallization of the TLR2/1 complex with a triacylated LP (Jin et al., 2007). Here it was shown that TLR2 contains two lipid-binding pockets, while TLR1 contains one enabling the heterodimer to connect with the three lipid chains of the triacylated LP. TLR6 lacking a similar pocket forms a heterodimer with TLR2 detecting diacylated LPs, as present, e.g. in Mycoplasma spp. (Takeuchi et al., 2001). Thus, triacylated spirochetal lipoproteins are recognized by a receptor complex including TLR2 and -1, which we could confirm in an overexpression system (Schro¨der et al., 2005).

LBP as binding partner for Osps The mammalian host has developed a number of humoral defense mechanisms in order to rapidly detect and respond to invading microorganisms. A serum protein with the potential to recognize cell wall compounds derived from Gram-negative and Grampositive microorganisms is LBP (Schumann et al., 1990). It is an acute-phase protein synthesized mainly in the liver, but also in the local epithelial cells of the lung, the gut and the oral cavity (Dentener et al., 2000; Ren et al., 2004; Vreugdenhil et al., 1999). We were able to show that LBP can also bind to different types of LPs in a plate-binding assay, as well as in a FACS-based assay (Schro¨der et al., 2004). Furthermore, LBP enhanced LP-induced cytokine induction in human monocytes. We have shown lately that LBP, while enhancing LPSinduced cytokine release when added in low concentrations, in high concentrations inhibited cytokine release (Lamping et al., 1998; Zweigner et al., 2001). This dualphase dose–response curve was not observed for LPs: high quantities of LBP also enhanced cytokine induction by LPs. For several of the cellular innate immune receptors, a strong species-specificity regarding the ligands recognized has been observed. For TLR4 it was shown that LPS precursors differed in their ability to block LPSeffects depending on the TLR4 species it bound to (Lien et al., 2000). Similarly, the specificity of certain oligonucleotide sequences to stimulate either human or murine cells was tightly connected to the respective TLR9 species (Bauer et al., 2001). We were interested whether human or murine cells differed in their ability to respond to LBP when added to LPs. As shown in Fig. 3 indeed there is a profound difference between the binding characteristics of human as compared to murine LBP towards synthetic LPs. Recombinant human LBP

ARTICLE IN PRESS N.W.J. Schro¨der et al. / Immunobiology 213 (2008) 329–340 0.8

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Fig. 3. Human, but not murine LBP binds to LPs. Microtiter plates were coated with triacylated or diacylated LPs (Pam3CysSK4 or Pam2CysSK4, respectively, EMC microcollections, Tu¨bingen, Germany) as well as LPS derived from E. coli O111:B4 or S. minnesota Re595 rough mutant (Sigma) at 30 mg/ml and 100 mM Na2CO3 (pH 8.2) for 6 h at room temperature. Blocking was performed with 150 mM NaCl and 50 mM HEPES (pH 7.4), supplemented with 10 mg/ml BSA (Roth) for 30 min at room temperature. Plates were then incubated with rhLBP (XOMA) or with recombinant murine LBP (Biometec, Greifswald, Germany) at increasing concentrations in the same buffer supplemented with 1 mg/ml BSA at room temperature for 2 h. Detection of bound LBP was performed using the anti-LBP Ab (1 mg/ml) and peroxidase-coupled streptavidin.

bound to plates coated with both diacylated and triacylated LPs (Pam3CysSK4 and Pam2CysSK4 purchased from EMC microcollections, Tu¨bingen, Germany) in a comparable fashion to smooth and rough LPS (preparations of Escherichia coli O111:B4 and S. minnesota Re595 rough mutant purchased from Sigma) in line with our previous findings (Schro¨der et al., 2003b). However, recombinant murine LBP (purchased from Biometec, Greifswald, Germany) tested at the same concentrations did not show significant interactions with both LPs, but

still bound to LPS (Fig. 3). Thus, human but not murine LBP acts as a PRR for bacterial LPs.

Evidence for an influence of TLR-mediated recognition of Borrelia on course of Lyme disease Genetic variations within the family of TLRs have been identified and correlations of the occurrence of single nucleotide polymorphisms (SNPs) and susceptibility to certain diseases have been described (Schro¨der

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and Schumann, 2005b). We analyzed a functionally relevant SNP leading to a change of amino acid arginine to glutamic acid at position 753, occurring frequently among white Caucasians, although completely absent in African populations, for its frequency among individuals suffering from LD (Mockenhaupt et al., 2006; Schro¨der et al., 2003a). While no difference in the overall frequency was observed, this SNP was almost completely absent among the subgroup of individuals suffering from late stage LD (Schro¨der et al., 2005). So, apparently a dysfunctional TLR2 receptor protects from the late inflammatory complications of LD. Furthermore, we could show that individuals heterozygous for this SNP reacted significantly weaker to an ex vivo challenge with Borrelia lysates. Macrophages from heterozygous mice crossbred from TLR2 k.o. and control mice also showed a significantly lessened response to Borrelia lysates (Schro¨der et al., 2005). This TLR2 SNP potentially plays a role in other inflammatory and cardiovascular diseases as we and others have shown (Hamann et al., 2005; Pierik et al., 2006). Recently we were able to show that genetic variations in TLR1, the heterodimerization partner of TLR2 for the recognition of triacylated LPs, are functionally relevant, and lead to suppression of extracellular expression of the TLR2/1 receptor complex (Johnson et al., 2007; Schumann and Tapping, 2007). This likely will lead to differences in susceptibility and course of disease and investigations regarding LD are currently underway.

Glycolipids in Treponema Chemical structure The presence of glycolipids in treponemes resembling bacterial LPS was repeatedly postulated since the early seventies (Jackson and Zey, 1973), but the first detailed report on the structure of a glycolipid in Treponema was published by Schultz et al. (1998). Here, Tde, a pathogen repeatedly associated with periodontitis, was extracted with phenol/chloroform/petrol ether (PCP), yielding a molecule of about 4 kDa in size. Chemical analysis revealed the presence of fatty acids and carbohydrates, in line with the overall structure of a glycolipid (Schultz et al., 1998). However, heptose, 3-deoxy-D-manno2-otulosonic acid (Kdo) and b-hydroxy fatty acids were absent, indicating a structure strikingly different from LPS. Cleavage by HF yielded a molecule of about 2.3 kDa, and data obtained by mass spectroscopy indicated the presence of a diacylglycerol lipid anchor plus a small core region consisting of hexoses and hexosamines (Schultz et al., 1998). Based on these studies, we conducted a comparative analysis of two treponeme strains which had been isolated just recently and characterized, T. maltophilum

(Tma) (Wyss et al., 1996), and T. brennaborense (Tbr) (Schrank et al., 1999) associated with periodontitis in humans and digital dermatitis in cattle, respectively (Moter et al., 1998; Schrank et al., 1999). Here, PCPextraction yielded only very little material, but substantial amounts could be recovered using a hot phenol/ water procedure (Schro¨der et al., 2000). Hexoses and aminohexoses were identified, with glucose dominating in Tbr and glucose, galactose and galactosamine dominating in Tma, respectively. Fatty acids were also recovered, indicating the presence of a glycolipid. However, in line with data published on Tde, heptose, Kdo and b-hydroxy fatty acids were absent. Compositional analysis revealed the presence of a diacylglycerol lipid anchor in the glycolipid derived from Tma, resembling the outer membrane glycolipid isolated from Tde (Schro¨der et al., 2000; Schultz et al., 1998). In contrast to Tde, electrophoresis and silver staining of glycolipids derived from Tbr and Tma revealed the presence of several ‘‘repeating units’’, putatively consisting of approximately 5 hexoses for Tbr and approximately 20–30 hexoses for Tma, respectively. Additionally, the core region linking the lipid anchor to the repeating units could be identified as a trisaccharide consisting of hexose–hexosamine–hexose (Schro¨der et al., 2000). A following report investigating a phenol/ water extract of T. medium identified a glycolipid containing a diacylglycerol lipid anchor as well, and compositional analysis of the polysaccharide part led to the identification of tetrasaccharide repeating units (Hashimoto et al., 2003). Taken together, the presence of diacylglycerol-containing glycolipids has been published for four different treponeme strains, and structural elucidation of these compounds, though yet incomplete, revealed striking similarities to lipoteichoic acid (LTA) from Gram-positive bacteria.

Interaction with the innate immune system Since both Tbr and Tma are associated clinically with severe inflammatory diseases, we performed an extensive analysis of biological characteristics of the isolated glycolipids from both strains (Opitz et al., 2001; Schro¨der et al., 2000, 2001). Both compounds induced the release of pro-inflammatory cytokines, in particular TNF, in both human and murine macrophages (Schro¨der et al., 2000, 2001). Cellular activation by both compounds was mediated by TLR2 and in the case of Tbr also partially by TLR4 (Opitz et al., 2001; Schro¨der et al., 2000). In human monocytes blocking of CD14 inhibited the release of cytokines induced by Tbr and Tma, and addition of LBP strongly enhanced cytokine release (Schro¨der et al., 2000). Interaction of the treponeme compounds with LBP could be demonstrated by binding assays as well, a feature that could be

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demonstrated for LTA derived from Gram-positive bacteria Streptococcus pneumoniae and Staphylococcus aureus also (Schro¨der et al., 2003b). In particular, LTA from Gram-positive bacteria and treponeme glycolipids, which resemble LTA, interacted with the same binding site in LBP as revealed by competitive binding studies (Schro¨der et al., 2001, 2003b). Furthermore, treponeme glycolipids also interacted with Polymyxin B and Endotoxin neutralizing Protein (ENP) isolated from Limulus polyphemus. Taken together, these studies not only clarified the mechanisms by which treponemes might activate the innate immune system, but also extended the array of ligands known to interact with LBP, Polymyxin B and ENP, three molecules formerly thought to specifically bind LPS. An interaction with LBP and CD14 could be demonstrated for glycolipids derived from T. medium and for a less characterized phenol/water extract from T. socranskii as well (Asai et al., 2003b, 2005; Lee et al., 2006). However, these compounds, via interacting with LBP and CD14, apparently inhibited cytokine induction caused by other TLR-ligands (Asai et al., 2003a, b; Lee et al., 2006). These diverging features could be due to yet undefined structural differences between the lipid anchors and core regions of the glycolipids investigated, as has been shown for other amphiphilic ligands, such as LPs (Schromm et al., 2007; Spiller et al., 2007).

LPS in Leptospira Several reports published in the course of the last decades have assumed the presence of LPS in Leptospira interrogans (Adler and Faine, 1982; Vinh et al., 1986). Leptospira exhibits several genes of the Lpx locus involved in LPS biosynthesis (Raetz and Whitfield, 2002), in contrast to Treponema and Borrelia, where no Lpx genes are found and where LPS has been convincingly ruled out. In line with this, L. interrogans features an LPS structurally related to LPS of Enterobacteriaceae (Que-Gewirth et al., 2004; Werts et al., 2001). However, the Lipid A portion of Leptospira LPS exhibits an unusual fatty acid composition and, most strikingly, a unique methylated phosphate residue (Que-Gewirth et al., 2004). Furthermore, in contrast to LPS of Enterobacteriaceae, leptospiral LPS preparations activated human immune cells via TLR2, not TLR4 (Werts et al., 2001). It has to be mentioned that activation of TLR2 has also been demonstrated repeatedly for LPS isolated from the periodontal pathogen Porphyromonas gingivalis (Hirschfeld et al., 2001; Pulendran et al., 2001), whereas chemically synthesized P. gingivalis Lipid A acts as a TLR4-ligand (Ogawa et al., 2007). Therefore, at this point, it cannot be ruled out that the TLR2mediated activity of leptospiral LPS-preparations is due to contaminating strong TLR2-ligands, such as LPs (Ogawa et al., 2007).

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Glycolipids in Borrelia Low molecular weight glycolipids in Borrelia As mentioned above, LPS has been repeatedly proposed in Borrelia, but has meanwhile been ruled out after the whole genome of B. burgdorferi has been sequenced (Fraser et al., 1997). However, there was still evidence that glycolipids other than LPS may be present in these bacteria, in particular in B. burgdorferi (Honarvar et al., 1994), and a systematic analysis of glycolipids extracted from B. hermsii with chloroform/methanol revealed the presence of a-monogalactosyl-diacylglycerol (MGalD) as well as acylated and non-acylated cholesteryl-glucosides (Livermore et al., 1978). MGalD was later demonstrated in B. burgdorferi as well (Hossain et al., 2001). Based on our findings that treponemes exhibit glycolipids resembling LTA, we questioned whether compounds like these are also present in Borrelia. However, no complex glycolipids could be recovered from B. burgdorferi via extraction procedures based on phenol/water and butanol (Morath et al., 2001). We found several low molecular weight glycolipids, including MGalD (Schro¨der et al., 2003c) (Fig. 4). Additionally, acylated cholesteryl-galactoside (ACGal) and its non-acylated counterpart, cholesteryl-galactoside (CGal) were found (Schro¨der et al., 2003c) (Fig. 4). Thus, MGalD appears to be abundant in Borrelia, while cholesteryl-galactosides are specific for B. burgdorferi and cholesteryl-glucosides are exhibited by B. hermsi.

Interaction of MGalD with the innate immune system (NKT cells) We tested MGalD, ACGal and CGal for immunostimulatory properties employing HEK293 cells transfected with human TLR2 and TLR4 (Schro¨der et al., 2003c). Here, only CGal caused translocation of NF-kB in TLR2-transfected cells, but these results could not be reproduced with chemically deacylated ACGal, indicating that a contaminating TLR2-ligand, presumably lipoprotein, may be responsible for the activity of CGal (Schro¨der et al., 2003c). However, MGalD features some structural similarities with a-galactosylceramide (GalCer), a glycolipid originally isolated from a marine sponge known to activate natural killer T (NKT) cells via interaction with CD1d (Godfrey et al., 2004). Therefore, MGalD as well as synthetic derivatives were tested for activation of these cells, and were found to stimulate proliferation of NKT cells as well a secretion of cytokines, including IFN-g (Kinjo et al., 2006). In line with this, infection of mice with B. burgdorferi via tick bites led to a strong proliferation of NKT cells in vivo (Kinjo et al., 2006). These data define glycolipid antigens of B. burgdorferi as strong activators of innate immunity. However, the effect of recognition of

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Fig. 4. Glycolipids described in Borrelia. Shown is a thin-layer chromatography of a chloroform methanol extract of B. burgdorferi B31 as well as the chemical structure of the three low molecular weight glycolipids for which structure has been elucidated.

B. burgdorferi via NKT cells on the course of infection still needs to be elucidated.

ACGal as a candidate for vaccination approaches As stated above, ACGal and CGal are not biologically active regarding immediate cytokine release by immune cells. However, there is evidence that ACGal is an antigen frequently recognized during the course of LD, resulting in the formation of antibodies. Sera derived from patients suffering from LD were tested for the presence of anti-glycolipid antibodies, and 10 out of 12 sera were positive for ACGal, whereas only 2 reacted with MGalD and none with CGal (Schro¨der et al., 2003c). These studies were in line with a report showing that mice generate antibodies towards ACGal after intraperitoneal immunization (Ben-Menachem et al., 2003). Thus, ACGal appears to act as a strong glycolipid antigen leading to the formation of antibodies during LD. Meanwhile, chemical synthesis of ACGal coupled to serum albumin has been published and proposed as a potential vaccine against LD (Pozsgay and KublerKielb, 2007). However, the question whether antibodies raised against ACGal are important or dispensable for bacterial clearance, or whether they are involved in LD pathology, needs to be elucidated by future studies.

Conclusions There is ample evidence that spirochetal lipoproteins play an important role in innate responses of the host

towards members of the genera Treponema and Borrelia, and may serve as targets for vaccination against LD as well. The impact of innate immune recognition via TLRs could be demonstrated by several in vivo studies in mice and is also supported by data on the association of functional TLR-variants with LD. Furthermore, lipoeptides are recognized and transferred to their receptors by LBP in a species-specific manner. However, there is also an expanding amount of data available that outer membrane glycolipids may be important contributors to pathogenesis of spirochetal diseases, and in the case of ACGal in B. burgdorferi may act as interesting targets of future vaccination approaches. Leptospira differs significantly from Treponema and Borrelia by exhibiting LPS comparable to Enterobacteriaceae (Que-Gewirth et al., 2004). This finding may explain the fact that Leptospira, in contrast to other spirochetes, are able to survive outside the host for a considerable time, possibly due to the stronger permeation barrier formed by outer membrane LPS (Raetz and Whitfield, 2002). In contrast, treponemes do not possess LPS, but complex glycolipids linked to the outer membrane via a diacylglycerol lipid anchor. These molecules have meanwhile been described in five treponeme strains and share structural characteristics with LTA of Gram-positive bacteria. However, they differ regarding their capacity to induce cytokines via TLRs, the structural basis of which still needs to be identified. Finally, Borrelia do not bear complex glycolipids resembling LPS or LTA at all, but a number of low molecular weight glycolipids instead. To date, MGalD seems to be the only Borrelia glycolipid known to directly activate innate immune responses via NKT cells.

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Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (DFG) to N.W.J.S. and R.R.S. The project was part of the ‘‘Schwerpunkt Angeborene Immunita¨t’’ and was funded under the nos. Schr 726/1-1, Schr 726/1-2, and Schr 726/1-3. This review serves as final report (‘‘Abschlussbericht’’) of this project. We wish to thank Ulrich Za¨hringer, Division of Immunochemistry, Research Center Borstel, Germany, for his outstanding contributions to this project during numerous collaborations over the last years. Furthermore, the excellent technical assistance of Sabine Bobbe, Fra¨nzi Creutzburg and Diana Woellner, all Institute for Microbiology, Charite´, Berlin, is acknowledged.

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