FIGURE 4.

Replacement of EscV TMD5 and TMD6 by an alternative hydrophobic sequence abolishes T3SS activity. (A) Protein secretion profiles of EPEC WT, ΔescV, ΔescN and EPEC ΔescV strain carrying the pEscV_wt-His, pEscV-TMD5_ex-His, or pEscV-TMD6_ex-His plasmids grown under T3SS-inducing conditions with 0.1 mM IPTG. The secreted fractions were filtered and protein content was concentrated from the supernatants of bacterial cultures and analyzed by SDS-PAGE and Coomassie blue staining. The T3SS-secreted translocators and effectors EspA, EspB, EspD, NleA, and Tir are marked on the right of the gel. EspC, which is not secreted via the T3SS, is also indicated. EscV expression was examined by analyzing bacterial pellets by SDS-PAGE and western blot analysis with an anti-His antibody. Numbers on the left are molecular masses in kilodaltons. (B) Proteins extracted from HeLa cells infected with WT, ΔescN, ΔescV, or ΔescV carrying the pEscV_wt-His, pEscV-TMD5_ex-His, or pEscS-TMD6_ex-His, were subjected to western blot analysis using anti-JNK antibody and anti-actin (loading control). JNK and its degradation fragments are indicated.