HilD Induces Expression of Salmonella Pathogenicity Island 2 Genes by Displacing the Global Negative Regulator H-NS from ssrAB

Abstract

Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) have essential roles in the pathogenesis of Salmonella enterica. Previously, we reported transcriptional cross talk between SPI-1 and SPI-2 when the SPI-1 regulator HilD induces expression of the SsrA/B two-component system, the central positive regulator of SPI-2, during the growth of Salmonella to late stationary phase in LB rich medium. Here, we further define the mechanism of the HilD-mediated expression of ssrAB. Expression analysis of cat transcriptional fusions containing different regions of ssrAB revealed the presence of negative regulatory sequences located downstream of the ssrAB promoter. In the absence of these negative cis elements, ssrAB was expressed in a HilD-independent manner and was no longer repressed by the global regulator H-NS. Consistently, when the activity of H-NS was inactivated, the expression of ssrAB also became independent of HilD. Furthermore, electrophoretic mobility shift assays showed that both HilD and H-NS bind to the ssrAB region containing the repressing sequences. Moreover, HilD was able to displace H-NS bound to this region, whereas H-NS did not displace HilD. Our results support a model indicating that HilD displaces H-NS from a region downstream of the promoter of ssrAB by binding to sites overlapping or close to those sites bound by H-NS, which leads to the expression of ssrAB. Although the role of HilD as an antagonist of H-NS has been reported before for other genes, this is the first study showing that HilD is able to effectively displace H-NS from the promoter of one of its target genes.

In silico clustering of Salmonella global gene expression data reveals novel genes co-regulated with the SPI-1 virulence genes through HilD

“…Affinity-purified maltose-binding protein (MBP)-HilD, which is active in vivo and specifically binds to HilD-target genes in vitro 1429, and the DNA fragments contained in the respective transcriptional fusion of each gene, were used to perform electrophoretic mobility shift assays (EMSAs). As a positive control, a DNA fragment containing the regulatory region of hilA was also assessed.…”

“…For instance, the transcriptional regulator HilD, encoded in SPI-1, positively controls the expression of the genes within this island, as well as several other genes located outside SPI-1, which mediate Salmonella invasion of host cells430. Additionally, HilD also positively regulates several genes necessary for Salmonella replication inside host cells, including the SPI-2 genes41429.…”

“…Although HilD has a dominant role, the expression of HilA, and thus the SPI-1 genes, depends on a complex feed-forward positive regulatory loop formed by HilD, HilC and RtsA2326; both HilC and RtsA are AraC-like regulators and bind the DNA sequence recognized by HilD2728. HilD also induces directly, or indirectly through HilA, InvF or other regulators, the expression of several other virulence genes, including genes located in SPI-2, SPI-4, SPI-5 and other islands414293031323334. Furthermore, HilD directly controls the expression of the flhDC operon encoding FlhDC, the master transcriptional complex required for the expression of flagellar and chemotaxis genes3035.…”

The Hcp-like protein HilE inhibits homodimerization and DNA binding of the virulence-associated transcriptional regulator HilD in Salmonella

“…HilC and RtsA are AraC-like transcriptional regulators that bind the same DNA sequence recognized by HilD; HilC is encoded within SPI-1, whereas RtsA is encoded in another island. HilD also controls the expression of many other virulence genes located outside SPI-1, including acquired and ancestral genes, directly, or indirectly through HilA, InvF or other regulators (12,(17)(18)(19)(20)(21)(22)(23)(24)(25). In agreement with its role as a master transcriptional regulator for a high number of genes, the expression, concentration and activity of HilD is tightly controlled.…”

Integrated circuits: how transcriptional silencing and counter-silencing facilitate bacterial evolution

“…Notably, this activity is at least partly dependent on a direct interaction with the carboxyl-terminal domain of the RNAP α subunit (α-CTD) which is sensitive to the position of the HilC/D binding site [72, 73]. Whether this is true for HilC/D targets outside of SPI-1, such as ssrAB where HilD is thought to oppose H-NS [74, 75], remains to be determined.…”