Role of Nucleoid-Associated Proteins Hha and H-NS in Expression of Salmonella enterica Activators HilD, HilC, and RtsA Required for Cell Invasion

Abstract

The coordinate expression of Salmonella enterica invasion genes on Salmonella pathogenicity island 1 is under the control of the complex circuits of regulation that involve the AraC/XylS family transcriptional activators HilD, HilC, and RtsA and nucleoid-associated proteins. Single-copy transcription fusions were used to assess the effects of nucleoid-associated proteins Hha and H-NS on hilD, hilC, and rtsA expression. The data show that all three genes, hilD, hilC, and rtsA, were repressed by H-NS and/or Hha. The repression of rtsA was the highest among tested genes. The level of rtsA-lac was equally elevated in hns and hha mutants and was further enhanced in the hns hha double mutant under low-osmolarity conditions. Electrophoretic mobility shift experiments showed that H-NS and Hha directly bind to the rtsA promoter. In addition to the negative control that was exerted by H-NS/Hha under low-osmolarity conditions, the homologous virulence activators HilD, HilC, and RtsA (Hil activators) induced rtsA-lac expression in a high-salt medium. A DNase footprinting assay of the rtsA promoter revealed one common DNA-binding site for all three Hil activators centered at position ؊54 relative to the transcriptional start site. In the absence of Hha and H-NS, however, osmoregulation of the rtsA promoter was lost, and Hil activators were not required for rtsA transcription. These results taken together suggest that the HilD, HilC, and RtsA proteins induce the transcription of the rtsA promoter by counteracting H-NS/Hhamediated repression.

HilD-mediated transcriptional cross-talk between SPI-1 and SPI-2

“…Affinity-purified maltose-binding protein (MBP)-HilD and H-NS-His 6 were then used to perform electrophoretic mobility shift assays (EMSAs) with fragments encompassing different regulatory regions. In agreement with other reports (30,34,35), MBP-HilD shifted specifically DNA fragments containing the regulatory regions of hilA or hilD at a concentration of 0.1 M (Fig. 4A and Fig.…”

Structural Insights into the Regulation of Foreign Genes in Salmonella by the Hha/H-NS Complex

“…The notion that Hha and YdgT act through H-NS to modulate gene expression is supported by experimental evidence that Hha/H-NS complex formation is required for silencing of the hlyCABD operon in E. coli, which encodes the ␣-hemolysin toxin (28,31). Complicating this model are reports that Hha can independently bind to specific regulatory sequences within the hilA promoter from S. Typhimurium and the esp operon of enterohemorrhagic E. coli (22,23,32,33). Despite extensive characterization of Hha-like molecules for over 2 decades, a clear understanding of how these proteins influence gene expression and why certain H-NS-repressed loci require Hha-co-regulation whereas others do not is still lacking.…”

“…Unlike H-NS, Hha-like proteins are found exclusively among enteric bacteria, and thus several species, such as Vibrio cholera and Bordetella pertussis, that encode H-NS homologues lack an associated Hha protein. In Salmonella, hha mutants display mild growth defects and increased expression of genes encoded within SPI1, responsible for bacterial invasion of epithelial cells, as well as genes encoded within SPI2 required for intracellular survival (22)(23)(24). As a result, hha mutants exhibit an initial hyperinvasive phenotype in cell culture and are attenuated for virulence in competitive murine infection models (22,24).…”

New insights into transcriptional regulation by H-NS

“…Activation of the SPI1 rtsA gene only requires the AraC family activators HilC and HilD if both H-NS and Hha are present [64,65]. The SPI1-encoded transcriptional activator HilA not only promotes expression of SPI1 genes but also appears to counter H-NS silencing of the SPI4 pathogenicity island [66], which acts in concert with SPI1 during Salmonella interactions with the host intestinal mucosa [67].…”