Effect of Mlc on hilE expression of Salmonella pathogenicity island 1

Abstract

Mlc protein is a global regulator of Phosphoenolpyruvate:sugar phosphotransferase system that plays an important role in the sugar uptake system of the prokaryotes and acts as a repressor of the pts genes. In previous researches, the invasion of mlc mutant into epithelial cells was shown to be decreased compared to that of wild type. In the absence of mlc, the expression of hilD, hilA and invF, the regulatory genes of Salmonella pathogenicity island 1, was decreased. In this research, it was found that the expression of hilE encoding the protein interacts with HilD was increased in the absence of Mlc. hilE/mlc double mutant strain was constructed via P22 transduction and the level of hilD expression was found to be not different between hilE and hilE/mlc double mutant strain. The virulence of those two strains also showed no differences. Primer extension assays were performed with mlc mutant strain containing E. coli or Salmonella Mlc overexpression vectors. The regulation of hilE expression was restored in Salmonella Mlc overexpressing strain but not in E.coli Mlc overexpressing strain. These results indicate that there are some functional differences between E.coli and Salmonella Mlcs. In vitro Mlc binding to target hilE promoter region was determined using gel shift assay with purified recombinant Salmonella Mlc. The purified Mlc alone could not bind to the hilE promoter region but the Salmonella cell extract could help Mlc to bind to the target DNA. However, Mlc could not bind to the hilE promoter region when the cell extract of hns mutant was used. These results imply that Mlc needs other intracellular factors to bind the target hilE promoter region so that it can act as a negative regulator of hilE expression. Further studies are needed to elucidate the role of H-NS in hilE transcription regulation by Mlc.