SSL4 protein sequences from human and bovine strains show no signs of host adaptation. A To investigate potential host adaptation of the SSL4 protein, the protein sequences from 15 human and animal (non-bovine) strains and 12 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 SSL4 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 a low and high concentration of MALP-2 (3 ng/mL or 30 ng/mL), Pam2CSK4 (0.01 ng/mL or 0.1 ng/mL), or Pam3CSK4 (3 μg/mL or 30 μg/mL) in presence or absence of two different SSL4 variants (from NCTC 8325 and CC97) in a final concentration of 10 μg/mL. IL-8 production was measured in subsequent ELISA and data points represent the actual IL-8 production (OD450). The mean ± SD of at least three independent experiments is shown. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test to compare between groups. *p ≤ 0.05, **p ≤ 0.01, ns (non-significant).