HilE is required for synergistic activation of SPI-1 gene expression in Salmonella enterica serovar Typhimurium

Abstract

Background: Salmonella enterica serovar Typhimurium is an intestinal pathogen capable of infecting a wide range of animals. It initiates infection by invading intestinal epithelial cells using a type III secretion system encoded within Salmonella pathogenicity island 1 (SPI-1). The SPI-1 genes are regulated by multiple interacting transcription factors. The master regulator is HilD. HilE represses SPI-1 gene expression by binding HilD and preventing it from activating its target promoters. Previous work found that acetate and nutrients synergistically induce SPI-1 gene expression. In the present study, we investigated the role of HilE, nominally a repressor of SPI-1 gene expression, in mediating this response to acetate and nutrients.

Results: HilE is necessary for activation of SPI-1 gene expression by acetate and nutrients. In mutants lacking hilE, acetate and nutrients no longer increase SPI-1 gene expression but rather repress it. This puzzling response is not due to the BarA/SirA two component system, which governs the response to acetate. To identify the mechanism, we profiled gene expression using RNAseq in the wild type and a ΔhilE mutant under different growth conditions. Analysis of these data suggested that the Rcs system, which regulates gene expression in response to envelope stress, is involved. Consistent with this hypothesis, acetate and nutrients were able to induce SPI-1 gene expression in mutants lacking hilE and the Rcs system.

Conclusions: While the exact mechanism is unknown, these results demonstrate the HilE, nominally a repressor of SPI-1 gene expression, can also function as an activator under the growth conditions investigated. Collectively, these results provide new insights regarding SPI-1 gene regulation and demonstrate that HilE is more complex than initially envisioned.

Keywords: Acetate; Gene regulation; HilE; SPI-1; Salmonella.

Fig. 1

Acetate and yeast extract (YE) synergistically induce hilA expression in the wild type (a) but not a ΔhilE mutant (b). Expression was determined by measuring β-galactosidase activity using transcriptional fusions of the hilA promoter to LacZ during growth in TB medium with the specified amount of yeast extact added. Sodium acetate was added at a concentration of 10 mM. Results show the mean and standard deviation from three biological replicates

Fig. 2

Transcriptional changes associated with the expression of the SPI-1 gene in the wild type (WT) and ΔhilE mutant during growth in TB medium, TB medium supplemented with 1% yeast extract (YE), TB medium supplement with 10 mM sodium acetate (Ac), and TB medium supplemented with 1% yeast extract and 10 mM sodium acetate. RNAseq data was collected in triplicate for each condition

Fig. 3

Formate and yeast extract increase hilA expression in both the wild type (a) and ΔhilE mutant (b); propionate increases hilA expression in both the wild type (a) and ΔhilE mutant but the further addition of yeast extract (YE) reduces it. Expression was determined by measuring β-galactosidase activity using transcriptional fusions of the hilA promoter to LacZ during growth in TB medium with the specified amount of yeast extact added. Sodium formate and sodium propionate were added at concentrations of 10 mM. Results show the mean and standard deviation from three biological replicates

Fig. 4

Transcriptional changes associated with the expression of the rcs and wca gene in the wild type (WT) and ΔhilE mutant during growth in TB medium, TB medium supplemented with 1% yeast extract (YE), TB medium supplement with 10 mM sodium acetate (Ac), and TB medium supplemented with 1% yeast extract and 10 mM sodium acetate (YE/Ac). RNAseq data was collected in triplicate for each condition

Fig. 5

The Rcs system is necessary for repression of hilA expression by acetate and yeast extract in a ΔhilE mutant. The figures show hilA expression in ΔrcsBC mutant (A) and ΔrcsBC ΔhilE mutant. Expression was determined by measuring β-galactosidase activity using transcriptional fusions of the hilA promoter to LacZ during growth in TB medium with the specified amount of yeast extact added. Sodium acetate was added at a concentration of 10 mM. Results show the mean and standard deviation from three biological replicates

Fig. 6

Regulatory network controlling SPI-1 and flagellar gene expression. While the mechanism is unknown, one possibility is that HilE is repressing Rcs mediated repression of SPI-1 and flagellar gene expression. Not shown are repression of flagellar gene expression by the Rcs system and RtsB and activation of flagellar gene expression by HilD