Figure 11. The virS/virR two-component regulatory system controls the Caco-2 induced increase in transcription of cpb and pfoA.

Caco-2 cells were infected with CN3685 (WT), CPJV47, CPJV47(pJVR4), CPJV47(pJVRS3) or CPJV47(pTS405) for 1 h at 37°C. Bacteria-containing supernatants were obtained to isolate the RNA as described in Material and Methods. Panels A–C, RT-PCR reactions were then conducted with 50 ng of RNA and primers that amplify the genes, (A) cpb, (B) pfoA or (C) plc/cpa. Where indicated, retrotranscriptase (RT) was (+) or was not (−) added into the reaction tubes. Reactions containing DNA from the WT was included. A 100 bp ladder is shown at left, with location of 200 bp (A), 700 bp (B) or 300 bp (C) markers is noted. Shown are representative figures of at least three independent experiments. Panel (D), Quantitative RT-PCR was then performed with 20 ng of the indicated RNA from infected Caco-2 cell cultures and primers that amplified the pfoA, cpb or plc/cpa gen. Average C_T values were normalized to the housekeeping polC gene and the fold differences were calculated using the comparative C_T method (2^−ΔΔC_T) (Livak and Schmittgen, 2001). Values below each bar indicate the calculated fold change relative to the wt strain CN3685. Shown is a representative graphic of three independent experiments.