FIG 5.
The C terminus of EspD is required for type III secretion of EspD, and C-terminal epitope tagging inhibits EspD secretion. (A) EPEC WT, ΔescN, and ΔsepD strains containing the vector pCX341 or pCX341 expressing C-terminally FLAG-tagged full-length EspD, EspD1–336, and EspD1–368 were grown in DMEM under type III secretion-inducing conditions. Whole-cell lysates (WCL) and secreted proteins (SP) were analyzed in SDS-PAGE by Western blotting using mouse monoclonal antibodies against FLAG. (B) EPEC ΔespD, ΔescN ΔespD, and ΔsepD ΔespD strains carrying the vector pCX341 or pCX341 expressing untagged full-length EspD, EspD1–336, and EspD1–368 were subjected to type III secretion assays. Whole-cell lysates and secreted proteins were analyzed by Western blotting using rat polyclonal antibodies against EPEC EspD. EPEC WT was included as a positive control on the left. Note that a faint band cross-reacting with the antibodies is located underneath the full-length EspD band.