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

Abstract

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.

FIG. 1.

Expression profile of a hilE-lacZY reporter throughout the growth curves of E. coli and S. enterica serovar Typhimurium. Strains were grown with shaking overnight in Lennox broth and then reinoculated into 20 ml of Lennox broth. Samples were taken every 40 min after the cultures began actively growing. Levels of β-galactosidase activity were determined for each sample taken. Plasmid pMAB56 is a single-copy mini-F plasmid that carries 1,000 bp of the putative hilE promoter fused upstream of a promoterless lacZY gene. Experiments were performed at least three times.

FIG. 2.

Overexpression of fimYZ activates hilE-lacZY expression in E. coli. Strains were grown with shaking in Lennox broth to the late stationary phase. Each strain carried plasmid pMAB69, which is a low-copy-number hilE reporter carried on the pRW50 vector. Plasmid pISF182 has the fimYZ genes cloned into pACYC184 and expressed from the tetracycline promoter. Plasmids pISF187 (fimZ⁺ fimY) and pISF189 (fimZ fimY⁺) are identical to pISF182 except for the insertion of a transcriptional terminator (see Materials and Methods). E. coli carrying only pISF187 or only pISF189 was not induced for hilE-lacZY expression, indicating that both fimY and fimZ are necessary for the expression of hilE. Expression levels were determined by measuring β-galactosidase activity. Experiments were performed at least three times.

FIG. 3.

Overexpression of fimYZ in S. enterica serovar Typhimurium leads to activation of a hilE-lacZY reporter. SL1344 (wild type), SL1344/pISF182 (fimYZ overexpressing), and SL1344 fimZ-kan, each containing the hilE-lacZY reporter pMAB69, were grown with shaking in Lennox broth to the late stationary phase. Levels of expression were measured by β-galactosidase activity. Each assay was performed at least three times.

FIG. 4.

Regulatory influence of fimYZ on hilA-lacZY expression. Deletion of fimZ increased hilA-lacZY expression, while overexpression of fimYZ within S. enterica serovar Typhimurium repressed an hilA-lacZY reporter. A deletion of the hilE gene eliminated the effect of fimYZ overexpression on hilA expression. Cultures were grown to the late stationary phase in Lennox broth. Levels of lacZ activity were measured by the β-galactosidase assay. Each assay was performed in triplicate and repeated three times.

FIG. 5.

Overexpression of fimYZ leads to repression of invasion of HEp-2 cells. Strains were grown under low-oxygen and high-osmolarity conditions. The deletion of hilE restored S. enterica serovar Typhimurium invasiveness in HEp-2 cells even when fimYZ were overexpressed. Values are presented as a percentage of invasion by the wild-type strain, with the invasiveness of that strain being set to 100%.

FIG. 6.

FimYZ activation of the hilE promoter requires the presence of sequences in the hilE promoter in E. coli and S. enterica serovar Typhimurium. Expression values for the hilE-lacZY reporter plasmids in E. coli are shown in panel A. The E. coli strains DH12S/pMAB69, DH12S/pMAB69/pISF182, DH12S/pMAB102, and DH12S/pMAB102/pISF182 were grown with shaking overnight in Lennox broth. Expression values for hilE-lacZY reporter plasmids in S. enterica serovar Typhimurium are shown in panel B. The S. enterica serovar Typhimurium strains SL1344 ΔhilE/pMAB69, SL1344 ΔhilE/pMAB69/pISF182, SL1344 ΔhilE/pMAB102, and SL1344 ΔhilE/pMAB102/pISF182 were grown with shaking overnight in Lennox broth. Plasmid pMAB69 encodes the hilE-lacZY reporter. Plasmd pMAB102 encodes the hilE-lacZY reporter with putative FimYZ binding sites deleted from the promoter. Plasmid pISF182 encodes functional fimY and fimZ genes. β-Galactosidase assays were performed at least three times.

FIG. 7.

Mapping the hilE transcriptional start site in relation to the putative translation start site for hilE and the putative FimYZ binding site. The primer extension products from pMAB69 and pMAB102 were primed with hilE23 and run alongside dideoxy sequencing reactions (lanes marked CTAG). The 5′ end of the hilE mRNA appears to initiate at two positions. The first mRNA initiates at an adenosine 55 bp upstream of the putative ATG start codon. The second transcription start site initiates at an adenosine 148 bp upstream of the putative ATG start codon. This second transcript starts 54 bp downstream of the putative FimYZ binding sites.

FIG. 8.

Model for processing environmental signals that affect SPI-1 invasion gene expression. An environmental signal activates (or deactivates) FimYZ, which then activate (or deactivate) hilE transcription. Activation of fimYZ also leads to expression of type 1 fimbriae and an adhesive phenotype. The activation of hilE transcription produces HilE protein, which then leads to repression of hilA expression and subsequent expression of the other SPI-1 genes through its interaction with the HilD regulatory protein.