SSL3 protein sequences from human and bovine strains show no signs of host adaptation. A To investigate potential host adaptation of the SSL3 protein, the protein sequences from 19 human and animal (non-bovine) strains and 15 bovine strains, that represent the major lineages, were aligned and subsequently a maximum likelihood tree was produced in MEGA v6.05. Bovine strains are indicated in blue and non-bovine strains in black. B Shows the variation in SSL3 amino acids that were previously found to be essential for TLR2 inhibition. This region is highly conserved between all strains, with the exception of strains clustering with MRSA252, which was previously described to differ in this region [6, 8]. C HEK-TLR2 cells expressing human or bovine TLR2 were stimulated for 6 h with MALP-2 (100 ng/mL), Pam2CSK4 (10 ng/mL), or Pam3CSK4 (30 μg/mL) in presence or absence of three different SSL3 variants (from NCTC 8325, CC771, and CC97) in a final concentration of 10 μg/mL. IL-8 production was measured in ELISA. Data points represent the relative IL-8 production based on the situation without SSL3 present (set on 1.0 for both human and bovine cell lines) and the mean ± SD of three to four independent experiments is shown. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test to compare between groups. Statistical significance is indicated for the SSL3 treated cells as compared to the non-treated cells. ****p ≤ 0.0001, ns (non-significant).