Identification of Chromosomal Genes in Yersinia pestis that Influence Type III Secretion and Delivery of Yops into Target Cells
Figure 7
Trimethoprim inhibits the Yersinia TTSS.
A. HeLa cells were infected with KIM5 or CHI 30 (yscQ−) expressing YopE-Bla. Infections were carried out at the indicated MOI for three hours in the presence or absence of 500 µg/ml of trimethoprim. Following infection, cells were stained with CCF2-AM and visualized by microscopy to assess translocation of the YopE-Bla reporter. Blue fluorescence = YopE-Bla injection; green fluorescence = no injection. B. Y. pestis KIM8 and KIM8 Δ1234, carrying pMM207 (yopM expressed from native promoter), were subcultured into MM9 and grown at 26°C for 2 hours. Trimethoprim was then added at the indicated concentrations (0, 15, or 30 µg/ml) and the cultures were shifted to 37°C for 3 hours, followed by TCA precipitation of supernatant (S) and cell pellet (P) fractions and immunoblotting with antibodies against YopM, YopN, YopD, YopR (TTSS secreted proteins), YscD (TTSS machine), RpoA (cytoplasmic protein), Ail (outer membrane protein). Degradation products are not indicated since KIM8 and KIM8 Δ1234 lack Pla protease. C. Secretion assays and western blotting was performed as in Panel B using Y. pestis KIM8 carrying pAH83 (yopM expressed from nptII promoter).