Lta from s aureus

Mice were injected intraperitoneally (IP) with 125 µg of LTA from S. aureus (Sigma-Aldrich, St. Louis, MO, USA) in 50 µL of sterile saline. Lipopolysaccharide (LPS) from Escherichia coli serotype 0127: B8 (Sigma-Aldrich, St. Louis, MO, USA) was injected IP at 125 µg in 50 µL of sterile saline per mouse. Murine recombinant tumor necrosis factor-α (TNFα) was injected IP at 500 ng in 50 µL of sterile phosphate buffered saline per mouse (R&D Systems, Inc., Minneapolis, MN, USA). Control animals were injected IP with 50 µL clinical grade saline (0.9% NaCl). All IP injections were done on the right side. Four hours after IP injections of LPS, LTA, TNFα or saline, animals were prepared for IVM and the microcirculation underlying the parietal peritoneum was observed.

Purified LPS from the outer membrane of E. coli (Sigma, St. Louis, MO) or LTA from S. aureus (Sigma) was used to measure the possible interaction between paenipeptin C' and LPS or LTA according to previous studies [14 (link), 15 (link)]. A stock solution of LPS or LTA was prepared in sterilized water, at 1 mg/mL, and kept at − 20 °C. LPS was added to the P. aeruginosa ATCC 27853 cell suspension (10⁶ CFU/mL) at a final concentration of 0, 10, 25, or 100 μg/mL. Then paenipeptin C′ was added to a final concentration of 16 μg/mL. The mixtures were incubated at 37 °C for 1 h. The number of surviving cells after treatment was determined by spread-plating on tryptic soy agar. Polymyxin B which binds to LPS was used at 2 μg/mL as a positive control. Similarly, the effect of LTA at various concentrations (0, 10, 25, or 100 μg/mL) on the antibacterial activity of paenipeptin C′ at 32 μg/mL was tested against a Gram-positive bacterium, S. aureus ATCC 29213. The inoculum size, incubation conditions, and the methods for surviving cell determination were similar to the procedures as described above in the LPS binding experiment. Nisin, a cationic lantibiotic antimicrobial peptide with known binding ability to LTA [15 (link)], was used at 16 μg/mL as a positive control.

Man Rogosa Sharpe (MRS) was obtained from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). Octyl-Sepharose column CL-4B, DEAE-52 cellulose, fetal bovine serum (FBS), fluorescein and isothiocyanate (FITC), and Hoechst 33342 were purchased from Shanghai SolarBio Bioscience and Technology Co., Ltd. (Shanghai, China). The standard LTA from S. aureus was obtained from Sigma-Aldrich (L2515). RAW 264.7 macrophage and Caco-2 cells were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). The cell counting kit-8 (CCK-8) and penicillin-streptomycin solution (P/S) were obtained from Beyotime Biotechnology (Shanghai, China). ELISA Kits of TNF-α, IL-10, and IL-6 were purchased from R&D Systems Europe Ltd. (Abingdon, UK). Antibodies for β-actin, p44, p-p44, JNK, p-JNK, p65, and p-p65 were obtained from Cell Signaling Technology. Other reagents were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

The S. aureus RN4220^{41 (link)}, Newman and MSSA1^{42 (link)} strains were cultured in tryptic soy broth (TSB; Becton Dickinson and Co., Franklin, NJ, USA) overnight at 37 °C with shaking. Various kinds of serum were purchased from MilliporeSigma (St. Louis, MO, USA), heat-inactivated by treatment at 56 °C for 30 min and stored at −20 °C until use. rhApoA-I was purified as described below and the human ApoA-II was purchased from Calbiochem (Merck Millipore, Billerica, MA, USA). Mice (ICR and C57BL/6J) were obtained from and CLEA Japan, Inc. Apoa1 gene knockout mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA) and bred to the age of 8–12 weeks. Lysocin E was prepared as previously described^{9 (link)} and lysocin E derivatives were synthesised as previously reported^{10 (link)}. Lipids, such as PG from egg yolk lecithin, CL from bovine heart and LTA from S. aureus were purchased from MilliporeSigma. The other reagents were purchased from Fujifilm Wako Pure Chemicals (Tokyo, Japan) or MilliporeSigma except where noted.