Lipoteichoic acid

Mice. C57BL/6J specific pathogen free (SPF) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Gender matched mice between 8–12 weeks of age were used for each experiment.
Reagents and antibodies. Lipoteichoic acid (LTA, S. aureus origin), peptidoglycan (PGN, S. aureus origin), Pam3CSK4, sparstolonin B (SsnB), phorbol 12-myristate 13-acetate (PMA), ionomycin, ovalbumin (OVA) and α-galactosylceramide (α-GalCer) were all purchased from Sigma Aldrich (St Louis, MO, USA). Dispase and collagenase were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Percoll was purchased from GE Healthcare (Chicago, IL, US). The Cytofix/Cytoperm kit was purchased from BD Bioscience (Franklin Lakes, NJ, USA). Recombinant murine granulocyte macrophage-colony stimulating factor (rmGM-CSF) was purchased from Peprotech (Rocky Hill, NJ, USA). Anti-CD11c (N418), anti-CD11b (M1/70), anti-CD207 (4C7), anti-CD103 (2E7), anti-CCR7 (4B12), anti-CLA (HECA-452), anti-CD80 (16-010A1), anti-CD86 (GL-1), anti-CD45 (30-F11), anti-CD3 (17A2), anti-CD4 (GK1.5), anti-IL-17A (BL168), anti-CD16/CD32 (2.4G2) (93), and 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) were all purchased from Biolegend (San Diego, CA, USA). Anti-MHC-II (M5/114.15.2), anti-CD11b (M1/70), anti-TCRβ (H57–597), anti-ɤδTCR (GL3), anti-IFN-γ (XMG1.2), anti-IL-4 (8D4–8), and anti-Toll-like receptor 2 (TLR2) were all purchased from Thermo Fisher Scientific (Waltham, MA, USA). CD1d-tetramer was purchased from Proimmune (Oxford, UK). Anti-MHC class I (HB159) and anti-MHC class II (M5/114) was purchased from Bio X Cell (West Lebanon, NH, USA). The isotype-matched control for each antibody was purchased from the same company.
S. aureus culture. The frozen S. aureus (MRSA; USA300) stock was thawed on ice, then transferred to a tryptic soy broth (TSB; BD bioscience, Franklin Lakes, NJ, USA) and cultured at 37 °C for 18 hr with shaking. The colony forming units (CFU) were calculated in each culture. Heat-killed S. aureus (HK-SA) was prepared with heating at 95 °C for 30 min. The heated S. aureus suspension was centrifuged at 10,000 rpm for 1 min to harvest the bacteria cells, then the cell pellet was resuspended in phosphate buffered saline (PBS) or 0.9% NaCl.
Lipoprotein isolation and preparation of the cell wall component from S. aureus. Lipoprotein was isolated from S. aureus by following a method described in previous reports with modifications [18 (link),19 (link),23 (link)]. Briefly, cultured S. aureus (10^7–8 CFU/mL) was harvested by centrifuging at 5000× g for 20 min. The pellet was washed twice by 20 mM Tris-HCl (pH 8.0). The pellet was resuspended in 20 mM Tris–HCl (pH 8.0), then the bacterial cell was crushed with 0.3 mm stainless beads. The treated suspension was centrifuged at 5000× g for 20 min, then the supernatant was harvested as the protein suspension. The suspension was mixed with 100% ethanol and kept at −20 °C overnight. The sample was centrifuged at 12,000× g for 15 min, then the precipitated pellet was washed with 80% ethanol and centrifuged again at 12,000× g for 5 min. The precipitated pellet was dissolved with 1 M urea/50 mM Tris–HCl, 50 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0) (Crude Protein Extract; CPE). Triton X-114 was added to the protein suspension (final 1%), then the suspension was incubated at 4 °C with gentle mixing. The incubated suspension was heated at 37 °C, forming the micelle phase-containing lipoprotein. The micelle phase was extracted and lipoprotein (Clude S. aureus-lipoprotein; SA-LP) was harvested by following a method for CPE precipitation. The SA-LP was separated to each fraction (L1 to L4) in a size dependent manner by using a molecular weight cut-off filter (Amicon ultra; Darmstadt, Germany). For preparation of the cell wall extract (CWE), the twice-washed S. aureus pellet was resuspended in 20 mM Tris–HCl (pH 8.0). The suspension was kept at −80 °C for 30 min, then sonicated for 20 min. The suspension was centrifuged at 5000× g for 20 min, and the pellet was harvested as CWE.
Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and silver stain. The extracted protein solution was diluted with 5 × SDS sample buffer (2% SDS, 62.5 mM Tris–HCl (pH 6.8), 10% glycerol, 0.01% bromophenol blue, 50 mM dithiothreitol (DTT). The proteins, separated by SDS-PAGE, were visualized with a Silver Stain Kit (Thermo Fisher Scientific, Waltham, MA, USA). Whole staining procedure was followed with the manual.
Mouse primary cell isolation. Skin leukocytes were isolated by following a method described in a previous report with modification [47 (link)]. Briefly, the extracted ear was washed with tissue washing buffer (RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 100 U/mL penicillin, 100 mg/mL streptomycin) at 37 °C for 30 min with gentle shaking. The ear was separated into the ventral and dorsal sheets from the cartilage, and incubated at 4 °C overnight with dispase working solution (tissue washing buffer containing 0.25 mg/mL of dispase) to separate the epidermal and dermal sheets. These sheets were chopped with scissors, then incubated at 37 °C for 30 min in collagenase working solution (tissue washing buffer containing 1 mg/mL collagenase and 0.01% DNase). The digested ear pieces were passed through a 5 mL syringe with a 22 G needle to make single cell suspensions. Lymph node cells were prepared from skin-draining LN (dLN) by following a method described in a previous report [48 (link)]. Briefly, isolated dLN was crushed on a dish and suspended in cell culture medium. The cell suspension was filtered through a 70 μm cell strainer, then twice washed with cell culture medium (RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin). Splenocytes were obtained from the spleen by following a method described in a previous report [48 (link)]. Briefly, isolated spleen was crushed on a 70 μm cell strainer, and the cells were suspended in cell culture medium. After being washed once with the cell culture medium, the cells were further resuspended in an erythrocyte lysis solution (155 mM NH₄Cl, 10 mM KHCO₃, 1 mM Na-EDTA, and 17 mM Tris–HCl (pH 7.3)). After being washed twice with cell culture medium, the cells were used as splenocytes. Mouse bone marrow leukocytes were obtained from the tibia and femur. After extracting the tibia and femur, bone marrow leukocytes were flushed out with a syringe containing cell culture medium. The cell suspension was filtered through a 70 μm cell strainer and washed once with cell culture medium, then the cells were treated with the erythrocytes lysis solution. After lysis, the cells were washed twice with cell culture medium, and the cells were used as bone marrow leukocytes. Pan-naïve T cells were isolated from the splenocyte by using an EasySep Mouse Pan-Naïve T Cell Isolation Kit (Stemcell Technology; Vancouver, BC, Canada). Naïve CD4⁺ and CD8⁺T cells were isolated from the splenocyte by using a MagniSort mouse CD4 naïve T cell or mouse CD8 naïve T cell Enrichment kit (Thermo Fisher Scientific, Waltham, MA, USA), respectively. LN and splenic dendritic cells were isolated by using MagniSort Mouse CD11c Positive Selection kit (Thermo Fisher Scientific, Waltham, MA, USA). The whole procedure for the cell isolation kit was performed by following the manual.
Mouse BMDCs preparation. Mouse BMDCs were prepared by following a method described in a previous report [49 (link)]. At day 0, 2.0 × 10⁶ of bone marrow leukocytes were suspended in 10 mL of DC culture medium (cell culture medium containing 20 ng/mL of rmGM-CSF), and the cells were seeded on a 100 mm dish. At day 3, 10 mL of the fresh DC culture medium was added to the cultured cells. At days 6 and 8, half of the cultured medium was collected and centrifuged, then the cell pellets were resuspended in 10 mL of the fresh DC culture medium. The cell suspension was put back into the original plate. At day 10, cells were ready to use for each experiment.
BMDC stimulation assay. Naïve BMDCs were seeded on a 6-well plate with DC culture medium, then the cells were stimulated by the S. aureus-derived component, LTA (10 μg/mL), PGN (10 μg/mL), SA-LP (10 μg/mL or identical amounts with isolated from 10⁶ of S. aureus), CWE (identical amount with isolated from 10⁶ of S. aureus), CPE (10 μg/m or identical amount with isolated from 10⁶ of S. aureus) and live-SA (10⁶) and HK-SA (10⁶) for 24 hr. The TLR2 agonist Pam3CSK4 (500 ng/mL) and TLR4 agonist LPS (100 ng/mL) were used for the positive control. TLR2 was blocked by the anti-mouse TLR2 monoclonal antibody (mAB) (10 μg/mL) or inhibited by Ssn B (100 μM). The stimulated cells were analyzed by flow cytometry for the detection of activation markers (CD80, DC86 and MHC class II). Cultured medium was harvested for the measurement of cytokine production by ELISA.
Flow cytometry. Cell surface markers and intracellular cytokines were analyzed by a flow cytometer (FACScalibur and LSR-II; BD Biosciences, Franklin Lakes, NJ, USA) with the fluorochrome-conjugated monoclonal antibodies described in reagents and antibodies. The cells were initially incubated with FcR blocker (anti-CD16/32; 2.4G2) at 4 °C for 10 min. For surface marker staining, the cells were incubated with the antibody at 4 °C for 30 min. Intracelluler cytokine staining was performed by using a Cytofix/CytoPerm Kit (BD Biosciences, Franklin Lakes, NJ, USA) by following the manual. Briefly, the cells stained with the antibody for the surface marker were fixed and permilized. The cells were incubated with the antibody for cytokine staining at 4 °C for 30 min. The dead cells were excluded by forward scatter, side scatter, and propidium iodide gating. All data were analyzed by BD FACS Diva (BD bioscience, Franklin Lakes, NJ, USA) or FlowJo (Tree Star; Ashland, OR, USA).
Murine skin inflammation model. To establish the skin inflammation model, anesthetized mice were treated with the antigen by intradermal (ID) injection into the ear (Figure S2). The antigen was dissolved or diluted with 0.9% saline, then applied into the dorsal side of the ear. After 48 or 120 hr of the treatment, the mice were sacrificed and the treated ear and skin-dLN was excised for use in each analysis.
In vivo antigen tracking. For antigen tracking, FITC-labeled SA-LP and OVA or non-labeled OVA (10 μg in each) were ID-injected into the mouse ear. After 48 hr of the treatment, the skin dLN was extracted from the treated mice, then the isolated LN cells were analyzed by flow cytometry.
Ex vivo antigen re-stimulation. The skin leukocytes were isolated from the SA-LP ID-injected mice ears, then the cells were labeled by CFSE. The labeled cells were cocultured with stimulated splenic DCs (SA-LP 1 μg/mL or LPS 100 ng/mL + OVA 10 μg/mL) or naïve splenic DCs at 37 °C for 24 hr. The proliferated cells were analyzed by flow cytometry.
In vitro antigen presentation. Isolated primary DCs were stimulated with SA-LP (10 μg/mL) overnight. The stimulated or naive DCs were cocultured with pan-naïve T, naïve CD4⁺, or CD8⁺T cells for 72 hr. At the last 6 hr of the coculture, the proliferated cells were re-stimulated with 100 ng/mL of PMA, 1 μg/mL of ionomycin, and protein transportation was inhibited with Golgi stop (BD Bioscience). The proliferated cells were analyzed by flow cytometry.
In vitro MHC blocking assay. MHC class I and II molecules on the DCs were blocked with antibodies by following a method described in a previous report with modification [50 (link)]. Briefly, LN isolated DCs were pre-incubated with the blocking antibody for MHC class I or MHC class II (10 μg/mL in each) for 1 hr. An isotype antibody was also used for the control. Then, the cells were stimulated with SA-LP (10 μg/mL) overnight. The stimulated DCs were cocultured with splenic naïve CD4⁺ or CD8⁺T cells for 72 hr in the presence of the blocking antibody or isotype antibody. At the last 6 hr of the coculture, the proliferated cells were re-stimulated with 100 ng/mL of PMA, 1 μg/mL of ionomycin, and protein transportation was inhibited with Golgi stop (BD Bioscience). The cells were analyzed by flow cytometry.
SA-LP-activated dendritic cell transfer. The primary DCs were isolated from WT mouse LN, then the cells were labeled with CFSE and stimulated with SA-LP (10 μg/mL) at 37 °C overnight. The treated DCs were washed with cell culture medium three times, then the cells (2.0 × 10⁶) were transferred into WT mice ears by ID injection. After 48 and 120 hr of the treatment, the skin-dLN and ear were used for each analysis.
Cytokine measurement by Enzyme-Linked Immuno Sorbent Assay (ELISA). The cytokine (TNF-α, IL-12p40 and IL-6) produced from the stimulated cell was measured by using a Mouse ELISA kit (Thermo Fisher Scientific, Waltham, MA, USA) for each target. The whole procedure was performed by following the manual.
Statistical analyses. A Student’s t-test was used to analyze the data for significant differences. Values of * p < 0.05, ** p < 0.01, and *** p < 0.001 were regarded as significant.

Lipoteichoic acid and protein detection by Western blot was undertaken essentially as previously described (Gründling and Schneewind, 2007b ). In brief, for sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis of cell-associated LTA and His-tagged proteins, 1 ml of overnight culture was mixed with 0.5 ml of 0.1 mm glass beads and lysed by vortexing for 45 min in the cold. Glass beads were sedimented by centrifugation at 200 g for 1 min, and 0.5 ml of the resultant supernatant transferred to a fresh tube. Bacterial debris and LTA were sedimented by centrifugation at 17 000 g for 15 min and suspended in protein sample buffer containing 2% SDS normalized for OD₆₀₀; that is, samples from a culture with an OD₆₀₀ of 2 were suspended in 50 μl of sample buffer. Samples were boiled for 20 min, centrifuged at 17 000 g for 5 min and 10 μl of samples loaded onto SDS-PAA gels. To determine the amount of LTA shed into the culture medium, 500 μl of culture was first centrifuged at 17 000 g for 5 min to pellet bacteria. Culture supernatant (100 μl) was removed, mixed with 100 μl of 2× protein sample buffer, boiled for 30 min and insoluble material removed by centrifugation at 17 000 g for 5 min. Supernatant samples were normalized based on OD₆₀₀ of 2, in that 10 μl of a culture of OD₆₀₀ of 2 was loaded. To determine if the His-tagged proteins were shed into the supernatant, 1.4 ml of culture was centrifuged at 17 000 g for 10 min to pellet the bacteria. One millilitre of the supernatant was transferred to a new tube, mixed with 100 μl of 100% trichloroacetic acid (TCA), vortexed, incubated on ice for 1 h and centrifuged for 10 min at 17 000 g. The supernatant was aspirated and the TCA precipitated pellet was washed twice with 1 ml of ice-cold acetone. Between wash steps, samples were incubated on ice for 1 h and debris collected by centrifugation as described above. After the final centrifugation step, pellets were air dried and suspended in 2× protein sample buffer normalized for OD₆₀₀; that is, samples from a culture with an OD₆₀₀ of 2 were suspended in 100 μl of sample buffer. The samples were boiled for 30 min and 10 μl analysed by Western blot. LTA samples were routinely loaded onto 15% SDS-PAA gels and probed with polyglycerolphosphate-specific LTA antibody (Clone 55 from Hycult biotechnology) and HRP-conjugated anti-mouse IgG (Cell Signalling Technologies, USA) used at 1:2000 and 1:10 000 dilutions respectively. His-tagged protein samples were routinely loaded onto 10% SDS-PAA gels and probed with HRP-conjugated His-tag-specific antibody (Sigma) used at a 1:10 000 dilution and Western blots were developed by enhanced chemiluminesce (ECL). Western blots were performed with at least three independently grown cultures in at least two independent experiments and representative images are shown.

Referred to the proteomic results, six related proteins, lipoteichoic acid synthase (Q2G093), signal peptidase I (Q2FZT7), teichoic acids export ATP-binding protein TagH (Q2G2L1), lipoprotein (Q2G0V0), UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (Q2FWH4), and D-alanine-D-alanine ligase (Q2FWH3), were selected for further verifiable quantification for three samples from the azalomycin F group or the blank one, using the parallel reaction monitoring (PRM) technology. According to the similar procedure to proteome analysis, the peptides from tryptic hydrolysis were dissolved in 0.1% (v/v) formic acid and separated on an EASY-nLC 1000 UPLC system with a reversed phase analytical column. The gradient elution consisted of an increase from 7% to 25% solvent B in 40 min, 22% to 35% in 12 min, 35% to 80% in 4 min, and then holding at 80% for the last 4 min. The analysis process was achieved at a constant flow rate of 0.4 μL/min. The separated peptides were subjected to an NSI source followed by MS/MS analysis in a Q ExactiveTM Plus (Thermo Fisher Scientific, San Jose, CA, USA). The electrospray voltage applied was 2.0 kV. The full scan of m/z ranged from 400 to 1000, and intact peptides were detected in the Orbitrap at a resolution of 70,000. The peptides were then selected for MS/MS analysis using an NCE setting as 27, and the fragments were detected in the Orbitrap at a resolution of 17,500. The data-dependent procedure alternated between one MS scan followed by 20 MS/MS scans. The AGC was set at 3e⁶ for full MS and 1e⁵ for MS/MS, respectively. The maximum IT was set at 50 ms for full MS and 180 ms for MS/MS. The isolation window for MS/MS was set at 1.6 m/z.
The resulting MS data were processed using Skyline (v.3.6). Peptide parameters were set as trypsin (KR|P) for the enzyme, zero for the max missed cleavage, 7 to 25 residues for the peptide length, and alkylation on Cys for fixed modification. Transition parameters were set as 2 and 3 for precursor charges, 1 for ion charges, and b and y for ion types. The product ions were set from ion 3 to the last ion. The mass tolerance of ion match was set as 0.02 Da.