FIG 5.

InvS regulates the level of PrgH. (A) InvS upregulates prgH-gfp expression in Salmonella. The 5′ UTR along with the full ORFs of SpaO, InvA, PrgK, and PrgH were translationally fused to GFP. Plasmids expressing the GFP fusion proteins were cotransformed with plasmids expressing InvS or the vector control into the ΔinvS strain. The background strain eliminates the potential effect that can be caused by chromosomal invS. Bacterial isocitrate dehydrogenase (ICDH) was detected using anti-ICDH polyclonal antibodies as the loading control. The levels of GFP fusion proteins were determined by Western blotting with polyclonal anti-GFP antibodies. The arrow indicates the expression of PrgH-GFP. (B) Quantification of GFP fusion protein expression from three independent experiments. Values represent GFP fusion protein expression levels after normalization to the expression of ICDH. The P value was calculated using the Student t test. (C) The expression of prgH-gfp was decreased in the absence of InvS. pXG30-derived PrgH-GFP was transformed into the WT or ΔinvS strain. Western blot of the 2-fold dilution series showing the expression of prgH-gfp. (D) InvS does not regulate the transcriptional level of prgH. A promoterless lacZ gene was placed under the prgH promoter in either the wild-type Salmonella or the ΔinvS mutant strain. β-Galactosidase activity was measured as described in Materials and Methods. The Salmonella WT strain without lacZ was used as the negative control. The data shown were obtained from three independent experiments. Results are presented as the means in Miller units. Error bars indicate standard deviations. (E) Overexpression of prgH partially restores the ΔinvS mutant invasion defect. HeLa cells were infected with Salmonella for 15 min at an MOI of 10. Relative bacterial invasion was determined by the gentamicin protection assay. The data shown were obtained from three independent experiments. The P values were calculated using the Student t test.