IpaD acts as a TLR2 agonist. The reporter system is based on NIH3T3 cells stably transfected with the NF-κB–inducible reporter gene SEAP together with the corresponding combinations of human TLRs. The amount of SEAP upon TLR induction can be compared in cell supernatants by addition of its substrate pNPP and measurement of the product OD at 405 nm. (A) OD at 405 nm 30 min after substrate incubation with supernatants from treated cells. Cells were treated for 18 h with indicated concentrations of IpaD in comparison to negative, positive, and spiked controls. The TLR2-1 and 2–6 reporter cell lines are shown in comparison to the control SEAP cell line without TLRs. (B) Induction of the TLR2-1 reporter cell line. The OD at 405 nm is presented over time for selected concentrations of IpaD in comparison to positive (Pam3CSK4) and negative (NIH3T3 control cell line) controls. (C) Induction of the TLR2-6 reporter cell line. The OD at 405nm is presented over time for selected concentrations of IpaD in comparison to positive (FSL-1) and negative (NIH3T3 control cell line) controls. Two independent experiments were performed in duplicate and all data are presented as mean ± SEM. Statistically significant differences to the control SEAP cell line were determined by two-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.