Fig. S2.
The optimized T3SS injection reporter is more sensitive at detecting T3SS translocation than the previously described IpgD-based protein. (A) Supernatants of ipaD strains expressing TEM1 (bla) fused to the optimized T3SS injection reporter or to IpgD [pBAD-IpgD-bla (6)] were incubated with nitrocefin. Enzymatic activity was calculated based on measurement of A486nm. Data are from three independent experiments. (B–E) CCF2-AM–loaded Jurkat T lymphocytes were infected for 30 min at different MOIs with WT Shigella expressing OspD1sh-M31L-bla or pBAD-IpgD-bla, followed by 2 h of incubation in medium supplemented with gentamycin to kill extracellular bacteria. Upon treatment with cytochalasin D to prevent invasion, injected-only cells were detected by flow cytometry (B). Without cytochalasin D treatment, all targeted cells were detected by flow cytometry (C–E). Under this condition, the same proportion of cells was detected as targeted at low MOIs with both reporters (C). However, fluorescence intensity of the targeted cells indicated that they were significantly more injected by the optimized reporter (D, red) compared with pBAD-IpgD-bla (D, black), which was confirmed by quantification of the ratio of mean fluorescence intensity (MFI) of cleaved CCF2-AM over uncleaved CCF2-AM (E). Data are from three independent experiments. **P ≤ 0.01; ***P ≤ 0.001 (Student’s t test compared with pBAD-IpgD-bla).