Fig. 4. TLR11 recognizes specific features of TgPRF.

(a) Peritoneal macrophages from wild-type (WT) and TLR11−/−, mice were stimulated for 24 h with 3.0 μg/ml of either TgPRF, TgPRF purified from Sf9 insect cells, or 5 mM of CpG as a positive control. Macrophages were stimulated with 3.0 μg/ml of human cofilin as a negative control. (b) Peritoneal macrophages were stimulated with 3.0 μg/ml of either TgPRF, acidic loop deletion mutant TgPRF (ΔAL), β-hairpin deletion mutant TgPRF (ΔBH), double deletion mutant TgPRF (ΔALBH), a TgPRF mutant with P. falciparum β-hairpin (TgPRF +Pf BH), actin-binding surface TgPRF point mutants 2PM or 6PM and a peptide with the acidic loop and β-hairpin sequence (ALBH peptide). (c) Peritoneal macrophages were stimulated with TgPRF, a TgPRF mutant with the acidic loop from C. parvum (TgPRF +Cp AL), a TgPRF mutant with the acidic loop from P. falciparum (TgPRF +Pf AL) and a TgPRF acidic loop deletion mutant that, like S. cerevisiae profilin, has two glycines in place of the acidic loop (TgPRF +Sc AL). (d) Peritoneal macrophages were stimulated with S. cerevisiae profilin (ScPRF), a ScPRF mutant containing the T. gondii acidic loop (ScPRF +Tg AL), a ScPRF mutant containing the T. gondii β-hairpin (ScPRF +Tg BH), and a ScPRF mutant containing both the acidic loop and β-hairpin (ScPRF +Tg ALBH). After 24 h, supernatant was removed and analyzed for IL-12p40 levels by ELISA. Each bar represents the mean ± SD of triplicate measurements of three independent experiments. Statistical analysis was performed using the Student's t test where † indicates P < 0.01; ‡ indicates P > 0.05 arbitrary units. In the columns marked with an asterisk, the IL-12p40 concentration was below detectable levels. Together these data show that the parasite-specific motif in TgPRF is the key determinant for recognition by TLR11, with the acidic loop as the primary determinant and the β-hairpin as a secondary but necessary determinant.