HilE interacts with HilD and negatively regulates hilA transcription and expression of the Salmonella enterica serovar Typhimurium invasive phenotype.

The ability of Salmonella enterica serovar Typhimurium to traverse the intestinal mucosa of a host is an important step in its ability to initiate gastrointestinal disease. The majority of the genes required for this invasive characteristic are encoded on Salmonella pathogenicity island 1 (SPI1), and their expression is controlled by the transcriptional activator HilA, a member of the OmpR/ToxR family of proteins. A variety of genes (hilC, hilD, fis, sirA/barA, csrAB, phoB, fadD, envZ/ompR, fliZ, hilE, ams, lon, pag, and hha) have been identified that exert positive or negative effects on hilA expression, although the mechanisms by which these gene products function remain relatively unclear. Recent work indicates that the small DNA-binding protein, Hha, has a significant role in repressing hilA transcription and the invasive phenotype, particularly in response to osmolarity signals. We have characterized the Salmonella-specific gene, hilE, and found that it plays an important regulatory role in hilA transcription and invasion gene expression. Mutation of hilE causes derepression of hilA transcription, and overexpression of hilE superrepresses hilA expression and the invasive phenotype. Bacterial two-hybrid experiments indicate that the HilE protein interacts with HilD, suggesting a possible mechanism for HilE negative regulation of hilA gene expression and the Salmonella invasive phenotype. Finally, we have found that the hilE gene resides on a region of the serovar Typhimurium chromosome that has many characteristics of a pathogenicity island.

Baxter MA ，Fahlen TF ，Wilson RL ，Jones BD ... -  《-》

The fimYZ genes regulate Salmonella enterica Serovar Typhimurium invasion in addition to type 1 fimbrial expression and bacterial motility.

An important step in Salmonella enterica serovar Typhimurium virulence is the ability to invade the intestinal epithelium. The invasion process requires a large number of genes encoded on Salmonella pathogenicity island 1 (SPI-1) at centisome 63 as well as genes located in other positions throughout the chromosome. Expression of the invasive phenotype is tightly regulated by environmental cues that are processed by a complex regulatory scheme. A central player in the invasion regulatory pathway is the HilA protein, which is transcriptional activator belonging to the OmpR/ToxR family. A number of positive regulators (hilC, hilD, fis, sirA/barA, csrAB, phoBR, fadD, envZ/ompR, and fliZ) and negative regulators (hha, hilE, lon, ams, phoPc and pag) have been identified that are able to alter expression of hilA transcription. Recent work has found that hilA transcription requires the HilD protein for activation. Other work has emphasized the importance of HilE as a negative regulator of hilA. Overexpression of hilE superrepresses hilA transcription, as well as the invasive phenotype. Two-hybrid experiments suggest that HilE exerts its regulatory influence on hilA through protein-protein interactions with HilD as the protein does not bind to the hilA promoter nor does it affect hilD transcription. As it seems likely that hilE plays an important role in translating environmental signals into invasion gene regulation, we have attempted to identify how the hilE gene itself is regulated. Our results indicate that the fimYZ genes, response regulatory proteins involved in type 1 fimbrial gene expression and recently implicated in motility gene regulation, are important activators of hilE expression. These findings indicate that invasion gene expression is coregulated with motility and adherence and provide experimental evidence that the expression of these virulence phenotypes is a subset of the overall regulation of bacterial physiology.

Baxter MA ，Jones BD 《-》

Roles of hilC and hilD in regulation of hilA expression in Salmonella enterica serovar Typhimurium.

Sequences between -332 and -39 upstream of the hilA promoter are required for repression of hilA. An unidentified repressor is thought to bind these upstream repressing sequences (URS) to inhibit hilA expression. Two AraC-like transcriptional regulators encoded on Salmonella pathogenicity island 1 (SPI1), HilC and HilD, bind to the URS to counteract the repression of hilA. The URS is required for regulation of hilA by osmolarity, oxygen, PhoP/PhoQ, and SirA/BarA. Here, we show that FadD, FliZ, PhoB, and EnvZ/OmpR also require the URS to regulate hilA. These environmental and regulatory factors may affect hilA expression by altering the expression or activity of HilC, HilD, or the unknown repressor. To begin investigating these possibilities, we tested the effects of environmental and regulatory factors on hilC and hilD expression. We also examined hilA regulation when hilC or hilD was disrupted or expressed to a high level. Although hilC is regulated by all environmental conditions and regulatory factors that modulate hilA expression, hilC is not required for the regulation of hilA by any conditions or factors except EnvZ/OmpR. In contrast, hilD is absolutely required for hilA expression, but environmental conditions and regulatory factors have little or no effect on hilD expression. We speculate that EnvZ/OmpR regulates hilA by altering the expression and/or activity of hilC, while all other regulatory conditions and mutations regulate hilA by modulating hilD posttranscriptionally. We also discuss models in which the regulation of hilA expression is mediated by modulation of the expression or activity of one or more repressors.

Lucas RL ，Lee CA 《-》

Transcription of the Salmonella invasion gene activator, hilA, requires HilD activation in the absence of negative regulators.

Salmonella enterica serovar Typhimurium causes human gastroenteritis and a systemic typhoid-like infection in mice. Infection is initiated by entry of the bacteria into intestinal epithelial cells and is mediated by a type III secretion system that is encoded by genes in Salmonella pathogenicity island 1. The expression of invasion genes is tightly regulated by environmental conditions such as oxygen and osmolarity, as well as by many bacterial factors. The hilA gene encodes an OmpR/ToxR family transcriptional regulator that activates the expression of invasion genes in response to both environmental and genetic regulatory factors. HilD is an AraC/XylS regulator that has been postulated to act as a derepressor of hilA expression that promotes transcription by interfering with repressor binding at the hilA promoter. Our research group has identified four genes (hilE, hha, pag, and ams) that negatively affect hilA transcription. Since the postulated function of HilD at the hilA promoter is to counteract the effects of repressors, we examined this model by measuring hilA::Tn5lacZY expression in strains containing negative regulator mutations in the presence or absence of functional HilD. Single negative regulator mutations caused significant derepression of hilA expression, and two or more negative regulator mutations led to very high level expression of hilA. However, in all strains tested, the absence of hilD resulted in low-level expression of hilA, suggesting that HilD is required for activation of hilA expression, whether or not negative regulators are present. We also observed that deletion of the HilD binding sites in the chromosomal hilA promoter severely decreased hilA expression. In addition, we found that a single point mutation at leucine 289 in the C-terminal domain of the alpha subunit of RNA polymerase leads to very low levels of hilA::Tn5lacZY expression, suggesting that HilD activates transcription of hilA by contacting and recruiting RNA polymerase to the hilA promoter.

Boddicker JD ，Knosp BM ，Jones BD 《-》

HilE Regulates HilD by Blocking DNA Binding in Salmonella enterica Serovar Typhimurium.

The Salmonella type three secretion system (T3SS), encoded in the Salmonella pathogenicity island 1 (SPI1) locus, mediates the invasion of the host intestinal epithelium. SPI1 expression is dependent upon three AraC-like regulators: HilD, HilC, and RtsA. These regulators act in a complex feed-forward loop to activate each other and hilA, which encodes the activator of the T3SS structural genes. HilD has been shown to be the major integration point of most signals known to activate the expression of the SPI1 T3SS, acting as a switch to control induction of the system. HilE is a negative regulator that acts upon HilD. Here we provide genetic and biochemical data showing that HilE specifically binds to HilD but not to HilC or RtsA. This protein-protein interaction blocks the ability of HilD to bind DNA as shown by both an in vivo reporter system and an in vitro gel shift assay. HilE does not affect HilD dimerization, nor does it control the stability of the HilD protein. We also investigated the role of HilE during the infection of mice using competition assays. Although deletion of hilE does not confer a phenotype, the hilE mutation does suppress the invasion defect conferred by loss of FliZ, which acts as a positive signal controlling HilD protein activity. Together, these data suggest that HilE functions to restrict low-level HilD activity, preventing premature activation of SPI1 until positive inputs reach a threshold required to fully induce the system.IMPORTANCESalmonella is a leading cause of gastrointestinal and systemic disease throughout the world. The SPI1 T3SS is required for Salmonella to induce inflammatory diarrhea and to gain access to underlying tissue. A complex regulatory network controls expression of SPI1 in response to numerous physiological inputs. Most of these signals impinge primarily on HilD translation or activity. The system is triggered when HilD activity crosses a threshold that allows efficient activation of its own promoter. This threshold is set by HilE, which binds to HilD to prevent the inevitable minor fluctuations in HilD activity from inappropriately activating the system. The circuit also serves as a paradigm for systems that must integrate numerous environmental parameters to control regulatory output.

Grenz JR ，Cott Chubiz JE ，Thaprawat P ，Slauch JM ... -  《-》