Identification of HilD-regulated genes in Salmonella enterica serovar Typhimurium

Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium) pathogenicity island 1 (SPI-1) encodes a type III secretion system required for invasion of host gut epithelial cells. Expression of SPI-1 virulence genes is controlled by a complex hierarchy of transcription factors encoded within and outside SPI-1. The master regulator of SPI-1, HilA, is itself regulated by three homologous transcription factors, HilD, HilC, and RtsA. HilD activates transcription of hilA and other target genes in response to environmental conditions associated with the intestinal microenvironment of the host. We have mapped the binding of HilD across the S. Typhimurium genome using chromatin immunoprecipitation-sequencing (ChIP-seq). Thus, we have identified 17 regions bound by HilD, including 11 novel targets. The majority of HilD targets are located outside SPI-1. We demonstrate transcription activation of 8 genes by HilD; four of these genes have not been previously described as being regulated by HilD, including lpxR, which encodes a lipid A deacylase important for immune evasion. We also show that HilD-activated genes are frequently activated by HilC and RtsA, indicating extensive overlap of the HilD, HilC, and RtsA regulons.

FIG 1

Sites of HilD association identified by ChIP-seq. (A) Validation of putative S. Typhimurium HilD target regions identified by ChIP-seq. Data are from ChIP of untagged 14028s or FLAG-tagged HilD, followed by quantitative real-time PCR. Occupancy units represent background-subtracted fold enrichment relative to a control genomic region within the sbcC gene. Error bars represent 1 standard deviation from the mean, based on three independent biological replicates, with the exception of lpxR/STM14_1613, for which only two replicates were performed. Asterisks indicate a significant difference between the occupancy unit values for tagged and untagged strains (**, P < 0.01; *, P < 0.02). (B) Comparison of ChIP-seq and ChIP-qPCR data for HilD-FLAG₃. Each data point represents a HilD target region identified by ChIP-seq and confirmed by ChIP-qPCR. ChIP-seq scores are the sum of the read counts on both strands at the highest-scoring position. ChIP-qPCR scores are in occupancy units.

FIG 2

β-Galactosidase assay showing HilD activation of target genes. β-Galactosidase assay of fusions of the indicated upstream regions fused translationally to a lacZ reporter gene on a single-copy plasmid. Assays were performed with E. coli strain AMD054 containing either empty pBAD24 plasmid or pBLP013 (hilD), under noninducing (no arabinose) or inducing (with arabinose) conditions, as indicated. Data are shown normalized to the values in the presence of pBLP013 (hilD) and arabinose. Error bars represent 1 standard deviation from the mean, based on at least three independent biological replicates.

FIG 3

β-Galactosidase assay showing activation of HilD target genes by HilC and RtsA. (A) Sequence alignment of predicted DNA-binding domains of HilD, HilC, and RtsA, shown in the CLUSTAL format (30). Shaded residues indicate those predicted to make base-specific contacts with DNA. (B) β-Galactosidase assay of fusions of the indicated upstream regions fused translationally to a lacZ reporter gene on a single-copy plasmid. Assays were performed with E. coli strain AMD054 containing pBLP011 (hilC), under noninducing (no arabinose) or inducing (with arabinose) conditions, as indicated. Data are shown normalized to the values for cells grown in the presence of arabinose. Error bars represent 1 standard deviation from the mean, based on at least three independent biological replicates. (C) As described above, but with pBLP010 (rtsA) used in place of pBLP011 (hilC).

FIG 4

Summary of HilD-bound regions and HilD-regulated genes. Shown are genes identified as being directly regulated by HilD and regions identified as being bound by HilD, based on this and previous studies. Genes given in gray text were not identified as being HilD bound in this study. Genes with gray shading have not previously been described as being HilD regulated or bound. The dashed box indicates genes (i) that are not regulated by HilD or for which regulation has not been tested and (ii) whose upstream region is bound by HilD or for which HilD binds within the gene. (Underlined text indicates intragenic binding.) Asterisks indicate HilD-associated regions for which regulation by HilD was not tested in this or previous studies.