Structural analysis and immunostimulatory potency of lipoteichoic acids isolated from three Streptococcus suis serotype 2 strains

Streptococcus suis serotype 2 is an important porcine and human pathogen. Lipoteichoic acid (LTA) from S. suis has been suggested to contribute to its virulence, and absence of d-alanylation from the S. suis LTA is associated with increased susceptibility to cationic antimicrobial peptides. Here, using high-resolution NMR spectroscopy and MS analyses, we characterized the LTA structures from three S. suis serotype 2 strains differing in virulence, sequence type (ST), and geographical origin. Our analyses revealed that these strains possess–in addition to the typical type I LTA present in other streptococci–a second, mixed-type series of LTA molecules of high complexity. We observed a ST-specific difference in the incorporation of glycosyl residues into these mixed-type LTAs. We found that strains P1/7 (ST1, high virulence) and SC84 (ST7, very high virulence) can attach a 1,2-linked α-d-Glcp residue as branching substituent to an α-d-Glcp that is 1,3-linked to glycerol phosphate moieties and that is not present in strain 89-1591 (ST25, intermediate virulence). In contrast, the latter strain could glycosylate its LTA at the glycerol O-2 position, which was not observed in the other two strains. Using LTA preparations from WT strains and from mutants with an inactivated prolipoprotein diacylglyceryl transferase, resulting in deficient lipoprotein acylation, we show that S. suis LTAs alone do not induce Toll-like receptor 2–dependent pro-inflammatory mediator production from dendritic cells. In summary, our study reveals an unexpected complexity of LTAs present in three S. suis serotype 2 strains differing in genetic background and virulence.

are considered low virulent and have been mostly associated with secondary infections and immunocompromised individuals (8,9). Consequently, we chose ST1, ST7, and ST25 strains for our analyses because of their importance as predominant virulent S. suis serotype 2 strains, which are responsible for most porcine and human infections worldwide (3).
Besides peptidoglycan and lipoproteins (LPs), wall teichoic acids and lipoteichoic acids (LTA) are the major constituents of the Gram-positive cell wall. LTA has been suggested to contribute to the virulence of S. suis, whereas the absence of D-alanine residues from its LTA has been associated with an increased susceptibility to the action of cationic antimicrobial peptides (10). In another study (11), LPs, which are often co-purified with LTA (12,13), were determined to be major activators of the porcine innate immune system. As of now, the detailed structure of the S. suis LTA remained elusive. In general, LTA contains a lipophilic anchor formed by diacylglycerol (DAG), which anchors these molecules to the cell membrane. The DAG is substituted with a glycosyl moiety at the O-3 position. These glycosyl moieties, as well as the attached complex backbone structures consisting of repetitive units (RUs), are highly variable between different species of Gram-positive bacteria. So far, five different types of LTA have been described, which are mainly characterized by the architecture of their RUs (polyglycerol phosphate (type I), complex glycosyl-glycerol-phosphate (type II ϩ III), glycosyl-ribitol-phosphate (type IV), and glycosyl-phosphate (type V)-containing polymers) (14).
We describe herein the structural analysis of LTA isolated from three S. suis serotype 2 strains of different background as representatives of the most clinically and epidemiologically important STs using chemical degradations, high-resolution MS analysis, as well as one-and two-dimensional, homo-and heteronuclear NMR spectroscopy. Finally, the immunostimulatory properties of these well-characterized LTA preparations were evaluated to understand their role in the activation and modulation of the host innate immune response by S. suis.

Results
For the structural analyses, LTA preparations from the three S. suis serotype 2 strains P1/7 (ST1), SC84 (ST7), and 89-1591 (ST25), as well as from their respective ⌬lgt mutants, were prepared according to our previously published workflow (15). The latter strains lack the gene encoding for the lipoprotein diacylglyceryl transferase (Lgt) and are therefore deficient in lipidation of prolipoproteins (16). In a first step, we compared the 1 H NMR spectra of native LTA from all three strains (Fig. 1). Spec-    Table 1. (# indicates the anomeric signal of a tiny amount of Glc I lacking Glc II.) Lipoteichoic acid of S. suis tra have been recorded in deuterated 25 mM sodium phosphate buffer, pH 5.5, at 300 K to suppress fast de-alanylation. In all three 1 H NMR spectra, the typical NMR signals for polyglycerol phosphate chains of type I LTA molecules (14,17) can be observed. Furthermore, all three strains are capable of modifying the glycerol O-2 position with alanine as indicated by the broad signal at ␦ H 5.43-5.35 for proton H-2 of the alaninesubstituted glycerol moieties, the signal at ␦ H 4.33-4.27 for the CH group, and the doublet at ␦ H 1.63 for the CH 3 group of alanine (17). Whereas the spectra of LTA from strains P1/7 and SC84 are almost identical, the spectrum of LTA from strain 89-1591 indicates a different binding position for some of the present glycosyl residues (Fig. 1). Therefore, the detailed analysis of the latter LTA will be described separately below. Notably, the 1 H NMR spectra of LTA from strains P1/7 and SC84 are virtually identical with the published 1 H NMR spectrum for LTA isolated from S. suis serotype 2 strain 31533 (10).
For a better characterization of the LTA structures, we generated defined part structures by hydrofluoric acid (HF) treatment, from which we obtained the respective lipid anchor and de-phosphorylated RUs. Afterward, we analyzed the interconnection of the RUs using de-O-acylated LTA, which was obtained by hydrazine treatment, using NMR and MS.

Preparation and structural analysis of the lipid anchor and LTA part structures from LTA of strains P1/7 and SC84
To elucidate the nature of the lipid anchor, we treated the isolated LTA of strains P1/7 and SC84 with 48% HF for 2 days at 4°C according to our previously published procedure (18). This

Structural analysis of hydrazine-treated LTA of strains P1/7 and SC84
Hydrazine treatment cleaves off all ester-bound residues like fatty acids and alanine and thus reduces the structural heterogeneity of LTA molecules. Therefore, the basic structural features of the LTA chain is accessible, because phosphodiester bonds remain intact (18 -20). In the following, the structural investigation of hydrazine-treated LTA (LTA N2H4 ) of S. suis strains P1/7 and SC84 by NMR is discussed. In Fig. 3, the 1 H, 13 C-heteronuclear single quantum correlation (HSQC) NMR of LTA N2H4 of strain P1/7 is depicted as an example for both strains, because almost identical spectra were obtained. The complete NMR chemical shift data are summarized in Table 1. LTA isolated from WT strains and their respective ⌬lgt mutants are structurally identical, and the respective 1 H NMR  Table 2. Relative abundance for a spectral region was always normalized to the respective base peak. Additional information about molecular assignments for higher molecular species are depicted in Fig. S7.

Lipoteichoic acid of S. suis
spectra of native LTA preparations from ⌬lgt mutants are depicted in Fig. S6 (for spectra of LTA preparations from WT strains see Fig. 1).
The MS analysis of LTA N2H4 of S. suis strains P1/7 and SC84 revealed a remarkably high diversity of LTA molecules. In total, we observed Ͼ60 different LTA moieties, which could be Table 2 Mass spectrometric analysis of de-O-acyl LTA of S. suis strains P1/7 (ST1) and SC84 (ST7) Summary of calculated and observed molecular masses (Da) for LTA preparations after hydrazine treatment. For each preparation, two independent MS analyses have been performed, and the identified molecules are listed as a combined list (A ϭ de-O-acyl glycolipid anchor (1b); RU X ϭ GroP; RU Y ϭ ␣-Glcp-(132)-␣-D-Glcp-(133)-GroP). Masses observed only in one of the two replicates are written in italic type. Accuracy of the measurement is stated as ⌬ppm; NDϭ not detected.

Molecule
Calculated mass Lipoteichoic acid of S. suis grouped into two major structural types. One series of molecules belonged to type I LTA with the observed de-O-acylated lipid anchor 1b ( Fig. 2) with different numbers (X ϭ 3-14) of glycerol phosphate (GroP)-repeating units. The second type of observed LTA contained, in addition, more complex RUs con- The number of GroP repeats (X) varied from 3 to 10, and 1 to 10 RUs of structure Y were present in these LTA N2H4 molecules. However, we only observed LTA molecules with 4 -9 GroP moieties, which had multiple repeats (more than one repeat) of structure Y attached. In Fig. 4A, a representative mass spectrum of the LTA N2H4 of strain P1/7 is depicted. In Fig. 4B, the region of 800 -2700 Da is enlarged, and peaks for observed LTA N2H4 molecules are assigned. The mass region containing molecules with higher molecular weight is shown in Fig. S7. The full list of identified LTA N2H4 molecules for S. suis strains P1/7 and SC84 is given in Table 2.
To further investigate the order of the different RUs, we selected different molecules for MS/MS experiments. As an example, the MS/MS spectrum for LTA N2H4 with X ϭ 5 and Y ϭ 3 (mass ϭ 2620.495 Da) is shown in Fig. 5. In this way, we could verify the consecutive order of the two different RU types X and Y. In Fig. 5A the complete overview of the MS/MS spectrum obtained in the negative ion mode is shown. Masses unequivocally presenting fragments occurring from a fragmentation starting from the de-O-acyl linker are labeled in blue in Fig. 5A, and the ones occurring from a fragmentation starting at the terminus are shown in red. Fragments that can exist from both cleavage directions are labeled black in Fig. 5A. In Fig. 5B, the observed fragment ions are assigned to the respective cleavage position in the molecule. A complete list of the observed fragments and their assignment is given in  Fig.  3). Integration of signals was done in 1 H NMR spectra obtained from LTA N2H4 of the WT and their respective ⌬lgt strains and have been averaged for the evaluation of RU ratios.  Table S4.

Lipoteichoic acid of S. suis Preparation and structural analysis of the lipid anchor and LTA part structures from LTA of strain 89-1591
The analysis of the lipid anchor and LTA part structures isolated from LTA of strain 89-1591 after HF treatment was done as described above. The glycolipid anchor of this strain was identified, as for the other two strains, as kojibiose-diacylglycerol (1a in Fig. 2). However, the observed molecules representing the dephosphorylated RUs of the LTA of strain 89-1591 differed significantly from the previously observed molecules. The major present molecule was identified as ␣-D-Glcp-(131),␣-D-Glcp-(132)-glycerol (5); 3 and ␣-D-Glcp-(132)glycerol (6) were observed as well. Besides that, small amounts of 2 (resulting from the completely de-O-acylated glycolipid anchor), 1-O-Ala-glycerol (7), as well as unbound glycerol (4; structures for all molecules are depicted in Fig. 2) and alanine are detectable in this preparation (Fig. S8). Molecule 7 is a result of the migration of the alanine moiety from O-2 to O-1 of the glycerol after phosphodiester bond cleavage (19), indicating the substitution of some Gro-P repeats with alanine on position O-2. The unbound alanine results from the decomposition of 7 during long-term NMR measurement into 4 and alanine. All NMR chemical shift data of 5, 6, and 7 (Fig. 2) are listed in Tables S5-S7.

Structural analysis of hydrazine-treated LTA of strain 89-1591
Hydrazine treatment of LTA isolated from S. suis strain 89-1591 was performed as for the other two strains. The 1 H, 13 C HSQC NMR of LTA N2H4 of strain 89-1591 is depicted in Fig. 6; the complete NMR chemical shift data are summarized in Table 3.
The MS analysis of LTA N2H4 of S. suis strain 89-1591 (Fig.  7A) revealed an even higher diversity of LTA molecules than observed for the other two investigated strains. As before, we observed one series of molecules that belonged to type I LTA with the observed de-O-acylated lipid anchor 1b carrying dif-ferent numbers (X ϭ 4 -20) of GroP repeats. In addition, more complex versions of these LTA molecules with additionally bound hexoses have also been observed. On the one hand, these can be ␣-D-Glcp residues 1,2-linked to GroP, which would lead to the de-phosphorylated RU molecule 6 (Fig. 2). On the other hand, these residues can be ␣-D-Glcp 1,3-linked to the GroP, leading to de-phosphorylated RU molecule 3 (Fig. 2). Finally, both glycosyl attachments can be present in the same RU, leading to de-phosphorylated RU molecule 5 (Fig. 2), which was the molecule with the highest abundance observed. Because of the multitude of combinatorial possibilities, especially with regard to the nonstoichiometric ␣-D-Glcp substituents of the glycerol O-2 position, which has the same additional mass as one glucose moiety within the LTA chain, the present LTA N2H4 molecules cannot be determined in such detail as for strains P1/7 and SC84. The mass spectrometric analysis depicted in Fig. 7A as well as two magnified sections of the spectrum (Fig. 7, B and C) show this increased complexity. The full list of identified LTA N2H4 molecules for S. suis strain 89-1591 can be found in Table S8. In total, we identified more than 165 different LTA moieties in the LTA N2H4 preparation of S. suis strain 89-1591, all of them measured with a mass deviation of Յ3.5 ppm. An evaluation of RU ratios as described above for LTA N2H4 from ST1/ST7 strains is not possible for LTA N2H4 of this strain, because the signal for H-2 of Gro has too much overlap with other signals in the 1 H NMR spectra.

Analysis of the fatty acid composition of S. suis LTA preparations
For the analysis of the fatty acid composition, the LTA preparations isolated from the ⌬lgt mutants have been used. These should give the most reliable values because most likely no other fatty acid-containing molecules are co-purified. As the most prominent fatty acid, hexadecanoic acid (16:0) was  Table 3.

Evaluation of the immunostimulatory properties of S. suis LTA preparations
The immunostimulatory properties of the different S. suis LTA preparations were characterized using murine bone marrow-derived DCs. DCs are innate immune cells known for their central role in the S. suis infection, including the production of pro-inflammatory mediators (21). The pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF), as well as the chemokines CXC motif chemokine ligand (CXCL) 1 and CC motif chemokine ligand (CCL) 3, were selected based on the fact that they are produced in important concentrations by DCs following S. suis infection (21). Significant levels of all four mediators were observed following activation of DCs with concentrations of 1, 3, 10, and 30 g/ml native LTA preparations from the three S. suis WT strains (Fig. S9). The observed levels of these mediators were very similar for LTA prepara-

Lipoteichoic acid of S. suis
tions from the three S. suis strains (P1/7, SC84, and 89-1591). Only for CCL3 was an increased level at 30 g/ml induced by LTA of strain 89-1591 observed. Moreover, levels of IL-6, CXCL1, TNF, and CCL3 were induced by all LTA preparations in a dose-dependent manner (Fig. S9).
LPs are often co-purified alongside LTA, and bacterial LPs of other Gram-positive pathogens are important activators of the innate immune response (22). Given the elevated levels of proinflammatory mediators produced by DCs following activation with the S. suis LTA preparations, the immunostimulatory properties of LTA preparations following treatment with H 2 O 2 was evaluated. H 2 O 2 oxidizes the thioether bond of LPs, which abolishes immunostimulatory activity of potentially co-purified LPs (13,23). H 2 O 2 -treated LTA preparations only induced little IL-6, CXCL1, TNF, or CCL3 production following activation with 3 or 30 g/ml, whereas nontreated LTA preparations induced significantly increased activity (Fig. 8), suggesting that co-purified LPs, but not the LTA, are important inducers of pro-inflammatory mediators by DCs, and this for all three strains of S. suis was evaluated.
To specifically determine the immunostimulatory potential of the LTA molecules themselves, without additive or synergis-tic effects of the pro-inflammatory LPs, LTA was prepared from Lgt-deficient mutants. Lgt is required for LPs to be biologically active and recognized by TLR2 (16,20). Accordingly, activation of DCs with LTA preparations from Lgt-deficient mutants of the three S. suis strains led to a complete abrogation of proinflammatory mediator production, regardless of the concentration of LTA used (p Ͻ 0.001) (Fig. 8).
Taken together, these results suggested that the co-purified LPs, but not the LTA, are the main activators of DCs when using LTA preparations from the S. suis strains P1/7, SC84, and 89-1591. Because LPs are recognized by TLR2 following dimerization with either TLR1 or TLR6, which allows us to discriminate between triacyl and diacyl motifs of LPs (24), DCs derived from WT and TLR2 Ϫ/Ϫ mice were used. In accordance with the above-mentioned results, TLR2 deficiency resulted in a complete abrogation of pro-inflammatory mediator production by DCs, and this is regardless of the LTA concentration used (3, 10, or 30 g/ml) (p Ͻ 0.001) (Fig. 9).

Discussion
This study provides the first detailed structural characterization of LTA isolated from the important porcine and opportu-  (1b; Fig. 2) and different numbers of GroP and Glc residues. The difference ⌬m ϭ ϩ8.05 Da corresponds to one GroP moiety less but one Glc moiety more in the overall composition. All identified molecules are listed in Table S8. Relative abundance for a spectral region was always normalized to the respective base peak.

Lipoteichoic acid of S. suis
nistic human pathogen S. suis. We investigated three different serotype 2 strains: P1/7 (ST1), SC84 (ST7), and 89-1591 (ST25). In preparation for further immunological studies, we constructed the respective ⌬lgt mutants, which are deficient in Lgtmediated prolipoprotein acylation (16,22), and we analyzed their LTA as well. For all strains, the LTA isolated from ⌬lgt mutants had the same chemical structure as the one isolated from the respective parental WT strain.
As the glycolipid anchor, we identified in LTA of all three strains kojibiose-diacylglycerol, a glycolipid anchor that has also been found in other streptococci and closely related species like Lactococcus lactis (19,25). When combining NMR and MSbased data, we were able to show that S. suis strains P1/7 and SC84 produce an almost identical LTA, which is most likely the same as present in S. suis serotype 2 strain 31533 (which is also an ST1 strain) as judged from a published 1 H NMR (10). This is in line with the close relationship of ST1 and ST7 strains, because they both belong to the CC1 (7). Interestingly, these strains contained two different types of LTA. One series of LTA molecules represents a type I LTA carrying only polymeric GroP chains connected to the kojibiose-diacylglycerol lipid anchor, which are identical to those LTA molecules identified in L. lactis G121 (25), as well as Streptococcus uberis 233, Streptococcus dysgalactiae 2023, and Streptococcus agalactiae 0250 (19). The second series of observed LTA molecules comprises, in addition, more complex glycosyl residue-containing RUs consisting of ␣-D-Glcp-(132)-␣-D-Glcp-(133)-GroP. Similar RU constitutions are known from type II or type III LTA molecules (14). By MS/MS experiments, we could verify the consecutive order of the two different repeating unit types in these LTA molecules. In S. suis strains P1/7 and SC84, a defined subset of LTA molecules with 3-10 GroP repeats was found to be elongated with 1-10 repeats of the more complex glycosyl residue-containing units. To the best of our knowledge this is the first example of such a regulated synthesis of a mixed-type LTA.
In S. suis strain 89-1591, LTA molecules of two different types were also observed. The type I LTA is of the same structure as the one determined in the other two strains. The RUs of the more complex LTA type consist either of ␣-D-Glcp residues 1,2-linked to GroP, ␣-D-Glcp residues 1,3-linked to the GroP, or most prominently both of these glycosyl attachments to GroP are present in the same repeat. A multitude of combinatory possibilities, especially with regard to the nonstoichiometric ␣-D-Glcp substituents at the glycerol O-2 positions in these LTA molecules, makes it impossible at this stage to determine whether the order of GroP repeats and more complex RUs is as regular as in strains P1/7 and SC84. The summary of the identified LTA structures considering also identified fatty acids and the presence of the possible, nonstoichiometric alanine substi-  Fig. 10. It is important to note that strain 89-1591 is the ST25 of the CC25, which is genetically distinct from strains of CC1. This strain also possesses lower virulence than ST1 and ST7 strains (9).
In our investigation, we showed that the pro-inflammatory potency of S. suis LTA molecules themselves is quite low when tested for pro-inflammatory mediator production from DCs. H 2 O 2 treatment of LTA preparations as well as the use of LTA isolated from ⌬lgt strains lead to a complete abrogation of inflammatory activity independently of the strain, which can only be observed if LTA preparations of WT strains are used. This activation of DCs is totally TLR2-dependent and can therefore be ascribed to the LPs co-purified with the LTA. This is consistent with a study describing the LPs as the important activators of the swine innate immune system present in S. suis (11).
In summary, our study revealed an unexpected complexity of LTA molecules present in S. suis serotype 2 strains from different genetic and virulence backgrounds. In all investigated strains, two different kinds of LTA molecules have been identified, whereas an ST-specific difference with regard to the incorporation of glycosyl residues into the complex mixed-type LTA has been observed. Strains P1/7 and SC84 are able to attach an 1,2-linked ␣-D-Glcp residue as branching substituent to the ␣-D-Glcp 1,3-linked to the GroP. In strain 89-1591, an exclusive glycosylation at the glycerol O-2 position was observed. Just recently, the first enzyme required in the glycosylation process of this position in LTA of Listeria monocytogenes has been identified (26). The identification and analysis of respective homologous glycosyltransferases involved in such reactions in S. suis as well as the analysis of the impact on bacterial physiology and virulence are currently under investigation. This will further foster the recent achievements in the understanding of the biological role of teichoic acid glycosylation in Gram-positive bacteria (27).
Deletion of genes was confirmed by PCR and sequencing. The oligonucleotide primers used for the constructions are listed in Table S9. The growth of the different Lgt-deficient mutants was determined to be similar to that of their respective WT strains (data not shown).  and 89-1591 (ST25). From all three investigated strains, a type I LTA composed of kojibiose-diacylglycerol and polyglycerol phosphate chains was isolated. All strains are capable of modifying the glycerol O-2 position with alanine. Exclusively in strain 89-1591, a potential glycosylation at the glycerol O-2 position was additionally observed. In all strains, a second kind of LTA molecule has been identified, whereas ST-specific difference with regard to the incorporation of glycosyl residues into the complex mixed-type LTA has been observed. Strains P1/7 and SC84 are able to attach an 1,2-linked ␣-D-Glcp residue as branching substituent to the ␣-D-Glcp 1,3-linked to the GroP, whereas in strain 89-1591, this branching substituent is absent.

Chemical treatments of LTA
Hydrazine treatment (to yield de-O-acyl LTA) or HF treatment (to isolate the LTA glycolipid anchor and the dephosphorylated repeats) was performed following previously described procedures (18). Notably, de-O-acylated S. suis LTA has to be desalted by dialysis against water (MWCO: 500 -1000 Da) instead of performing a size-exclusion chromatography. To destroy the TLR2 activity caused by potentially co-purified LPs, LTA preparations were treated with 1% H 2 O 2 for 24 h at 37°C followed by dialysis as described previously (23).

Quantification of fatty acids
Fatty acids were extracted and quantified from LTA preparations of the ⌬lgt mutant strains following our earlier described procedure (18), but with n-pentadecanoic acid (15:0; Sigma) used as an internal standard. Reported data for fatty acid ratios are the mean of two independent hydrolyses of the same LTA batch, both measured as two technical replicates. Different isoforms for unsaturated fatty acids (16:1; 18:1) are reported as one sum value.

NMR spectroscopy
Deuterated solvents were purchased from Deutero GmbH (Kastellaun, Germany). NMR spectroscopic measurements were performed in D 2 O or deuterated 25 mM sodium phosphate buffer (pH 5.5; to suppress fast de-alanylation) at 300 K on a Bruker Avance III 700 MHz (equipped with an inverse 5-mm quadruple-resonance Z-grad cryoprobe). Acetone was used as an external standard for calibration of 1 H (␦ H ϭ 2.225) and 13 C (␦ C ϭ 30.89) NMR spectra (32), and 85% of phosphoric acid was used as an external standard for calibration of 31 P NMR spectra (␦ P ϭ 0.00). Analysis of glycolipid 1 was performed in CD 3 OD, and spectra were calibrated using the residual solvent peak (␦ H ϭ 3.31, ␦ C ϭ 49.0) (32). All data were acquired and processed by using Bruker TOPSPIN V 3.0 or higher. 1 H NMR assignments were confirmed by 2D 1 H, 1 H COSY and total correlation spectroscopy (TOCSY) experiments. 13 C NMR assignments were indicated by 2D 1 H, 13 C HSQC, based on the 1 H NMR assignments. Inter-residue connectivity and further evidence for 13 C assignment were obtained from 2D 1 H, 13 C heteronuclear multiple bond correlation and 1 H, 13 C HSQC-TOCSY. Connectivity of phosphate groups were assigned by 2D 1 H, 31 P HMQC and 1 H, 31 P HMQC-TOCSY.

Mass spectrometry
All mass spectrometric analyses were performed on a Q Exactive Plus (ThermoFisher Scientific, Bremen, Germany) using negative ion mode. LTA fractions were diluted to a final concentration of 0.03 mg/ml in propan-2-ol, water, 30 mM ammonium acetate (50:50:4, v/v/v), which was adjusted with acetic acid to pH 4.5. The HESI source was operated at Ϫ3 kV with a flow rate of 5 l/ml using nitrogen as sheath gas at 5 atomic units. Survey MS 1 and MS 2 spectra were acquired with a resolution of 288,000 full width at half-maximum at m/z 200. MS 2 analyses were performed using the Triversa Nanomate (Advion, Ithaca, NY) as ion source applying a spray voltage of Ϫ1.1 kV and back pressure of 1.0 p.s.i. Precursor ions were selected with isolation window width of 1.5 Da, and collisioninduced dissociation was performed with 20 normalized collision energies. Deconvoluted spectra were computed using Xtract module of Xcalibur 3.1 software (ThermoFisher Scientific, Bremen, Germany).

Generation of bone marrow-derived DCs and cell activation
Murine bone marrow-derived DCs were generated as described previously (21) from the femur and tibia of WT (C57BL/6J) or TLR2 Ϫ/Ϫ (B6.129-Tlr2tmKir/J) mice. Prior to activation, cells were seeded at 1 ϫ 10 6 cells/ml, and different concentrations of the LTA preparations or cell culture medium alone (negative control) were added. Cell supernatants were collected 24 h later for quantification of secreted IL-6, TNF, CXCL1, and CCL3 by sandwich ELISA using pair-matched antibodies (R&D Systems).