Figure 10. The virS/virR two-component regulatory system regulates the Caco-2 cell-induced increase in CPB secretion.

A) Inactivation of the virR gene. PCR was performed with primers that amplify (6) a ~590 bp product or (7) ~710 bp virR wt gene using DNA from CN3685 (WT), CPJV47 or CPJV47(pJVR4). A 100 bp ladder is shown at left and migration of the 600 bp marker is noted. B) CPB secretion. Caco-2 cell cultures were infected with the indicated strain for 2 h at 37°C. Sterile culture supernatants were obtained and analyzed by Western blot using a mouse monoclonal anti-CPB (migration of the 35 kDa CPB protein is noted at left and right of the blot). C) Caco-2 cells were infected with CN3685 (WT), CPJV47, CPJV47(pJVR4), CPJV47(pJVRS3) or CPJV47(pTS405) for 1 h. RNA (50 ng) isolated from bacteria was used as template in RT-PCR reactions with primers that amplify (top panel) the virR gene or (bottom panel) the housekeeping polC gene. Where indicated, retrotranscriptase (RT) was (+) or was not (−) added into the reaction tubes. Reactions containing DNA from the WT strain was included. A 100 bp ladder is shown at left and the location of 700 bp (top panel) and 300 bp (bottom panel) markers is noted. Figures shown are representatives of at least three independent experiments. D) Production of the VirR protein. Cytoplasmic proteins (20 µg) from CN3685 (WT), CPJV47 or CPJV47(pJVR3) were electrophoresed and transferred to nitrocellulose membranes. The membranes were probed by Western blot using an anti-VirR antibody. E) Schematic diagrams showing the virR antisense vector (pJVBM11) or the plasmids pJVR4, pJVRS3 and pTS405. Note that pJVBM11 has the virR gene in the antisense orientation with respect to the virR promoter. pJVR3 only encodes the CN3685 virR gene, pJVRS3 encodes the virS/virR operon from CN3685, and pTS405 encodes the virS/virR operon from Strain 13. K, KpnI; X, XbaI; H, HindIII, P, PstI.