FIG. 6.

An HPLC fraction containing the cytotoxic activity in EP stimulates an NF-κB-regulated reporter gene through TLR2. (A) S. flexneri TCA-endotoxin and EP, but not PW-LPS, activate TLR2 signaling. 293 cells were transiently transfected with expression plasmids encoding TLR2 and an NF-κB-regulated luciferase reporter gene. The cells were stimulated with S. flexneri TCA-endotoxin (open circles), EP (closed circles), or PW-LPS (closed squares), and reporter gene activity was assayed. (B) The cytotoxic activity in EP was purified sequentially by preparative SDS-PAGE and RP-HPLC. RP-HPLC fractions were assayed for cytotoxic activity on THP-1 cells, and an active fraction was identified as described in Materials and Methods. 293TLR2/CD14 cells were transiently transfected with the NF-κB-regulated luciferase reporter construct as described previously (1) and stimulated with the indicated dilutions of the cytotoxic HPLC fraction (HPLC Active) or an identical HPLC fraction collected from a blank HPLC run (HPLC Blank). As a positive control for TLR2 activation, cells were stimulated with sBLP. (C) The indicated cell lines were transiently transfected with an NF-κB-regulated luciferase reporter construct and incubated with 1:2,000 (black bars), 1:20,000 (gray bars), or 1:200,000 (white bars) dilutions of the cytotoxic HPLC fraction.