OmpR regulates the two-component system SsrA-ssrB in Salmonella pathogenicity island 2 - PubMed (original) (raw)

OmpR regulates the two-component system SsrA-ssrB in Salmonella pathogenicity island 2

A K Lee et al. J Bacteriol. 2000 Feb.

Abstract

Salmonella pathogenicity island 2 (SPI-2) encodes a putative, two-component regulatory system, SsrA-SsrB, which regulates a type III secretion system needed for replication inside macrophages and systemic infection in mice. The sensor and regulator homologs, ssrAB (spiR), and genes within the secretion system, including the structural gene ssaH, are transcribed after Salmonella enters host cells. We have studied the transcriptional regulation of ssrAB and the secretion system by using gfp fusions to the ssrA and ssaH promoters. We found that early transcription of ssrA, after entry into macrophages, is most efficient in the presence of OmpR. An ompR mutant strain does not exhibit replication within cultured macrophages. Furthermore, footprint analysis shows that purified OmpR protein binds directly to the ssrA promoter region. We also show that minimal medium, pH 4.5, induces SPI-2 gene expression in wild-type but not ompR mutant strains. We conclude that the type III secretion system of SPI-2 is regulated by OmpR, which activates expression of ssrA soon after Salmonella enters the macrophage.

PubMed Disclaimer

Figures

FIG. 1

ssrA gene expression is reduced significantly in an ompR mutant background. RAW264.7 cells were infected for 2 h with strains bearing reporter plasmids. The histograms show GFP expression from extracellular bacteria (thin lines) and intracellular bacteria (thick lines). Peak fluorescence intensity is indicated for each sample. (a) Macrophages were infected with the following strains: SL1344 (wild type) with pMIC-10C32 (ssrA-gfp), P3F4 (ssrA mutant) with pMIC-10C32, PhoP (phoP mutant) with pMIC-10C32, and SL1344K (rpoS mutant) with pMIC-10C32. (b) Macrophages were infected with SL1344 with pMIC-10C32, CJD359 (ompR mutant) with pMIC-10C32, SL1344 with pFMI10 (ssaH gfp), and CJD359 with pFMI10. The experiments shown in panels a and b were performed on different days.

FIG. 2

Kinetics of induction for _ssrA_- and ssaH-gfp fusions inside macrophages. RAW264.7 macrophages were infected with the following bacteria: SL1344 (wild type) with pMIC-10C32 (ssrA-gfp), SL1344 with pFMI10 (ssaH-gfp), CJD359 (ompR mutant) with pMIC-10C32, or CJD359 with pFMI10. The histograms show GFP expression from extracellular bacteria (thin lines) and intracellular bacteria (thick lines) at each time point postinfection. The axes for all histograms are identical.

FIG. 3

Comparison of survival and replication in RAW264.7 macrophages. RAW264.7 macrophages were infected with either SL1344 (wild type), PhoP (phoP), P3F4 (ssrA), CJD359 (ompR), or P3F4-CJD359 double mutant (ssrA/ompR) for 1 h before the addition of gentamicin. Gentamicin-protected CFU were determined at 3 (solid bars) and 23 (hatched bars) h postinfection.

FIG. 4

OmpR protein binds the ssrA promoter region. A 335-bp fragment spanning the ssrA promoter region was the target in a DNase protection assay. Purified OmpR protein binds the region −207 through −231 from the ATG of the ssrA gene. Each lane contained target DNA plus the indicated proteins. The exact sequence of the binding region is indicated, with protected sequences in boldface. The italicized letters indicate a potential OmpR binding half-site that cannot be determined from this experiment.

FIG. 5

Effects of ssrA promoter region deletions on expression inside macrophages. Different fragments of the ssrA promoter region cloned upstream of gfp in plasmid pFPV25 are shown. RAW264.7 cells were infected with wild-type bacteria harboring the indicated plasmids. The histograms show GFP expression from extracellular bacteria (thin lines) and intracellular bacteria (thick lines). The vertical line drawn through the histograms shows the peak fluorescence of extracellular bacteria from constructs pMIC-10C32 and pANT30.3. Peak fluorescences are indicated for each sample. Maps are not drawn to scale.

FIG. 6

Minimal medium at low pH is optimal for in vitro induction of ssrA and ssaH transcription. Salmonella cells were grown in LB broth overnight and subcultured in the indicated minimal media for 3 h. The histograms show GFP expression from bacteria carrying plasmids pFMI10 (ssaH-gfp) subcultured in LB broth (thin lines), pMIC-10C32 (ssrA-gfp) subcultured in minimal medium (dotted lines), or pFMI10 subcultured in minimal medium (thick lines). Peak fluorescences are indicated for each population. The histograms for bacteria with pMIC-10C32 subcultured in LB broth are superimposed on histograms from bacteria carrying plasmid pFMI10 subcultured in LB broth (data not shown).

FIG. 7

Increasing the osmolarity of minimal medium at pH 4.5 in an OmpR+ strain represses ssrA and ssaH expression. Salmonella was grown in LB broth overnight and subcultured in minimal medium at pH 4.5 (thin lines) or minimal medium at pH 4.5 plus 0.5 M NaCl (thick lines). The histograms show GFP expression from the indicated strains subcultured in either low (thin lines)- or high (thick lines)-osmolarity media. Peak fluorescences are indicated for each population. Bacteria subcultured in LB broth did not express significant levels of GFP (data not shown).

FIG. 8

Model for Salmonella SPI-2 regulation inside host macrophages. The OmpR-EnvZ system responds to the intracellular environment, possibly stimulated by the acidic pH and low osmolarity of the phagosome. OmpR binds to the ssrA promoter region to activate transcription of the ssrAB genes. Later, SsrB detects a different environmental stimulus in the vacuole. SsrB activates expression of the type III secretion system encoded within SPI-2, which then allows for replication inside cells and systemic infection in mice. The solid arrows indicate supporting data from this study and others (12, 35, 36, 64, 74). OmpR and SsrB are hypothesized to be phosphorylated (as indicated by “P”) in this model. The shaded proteins and genes represent those studied here. The dashed arrows indicate speculative function based on protein homology, not experimental evidence.

References

1. Abshire K Z, Neidhardt F C. Analysis of proteins synthesized by Salmonella typhimurium during growth within a host macrophage. J Bacteriol. 1993;175:3734–3743. - PMC - PubMed
1. Alphen W V, Lugtenberg B. Influence of osmolarity of the growth medium on the outer membrane protein pattern of Escherichia coli. J Bacteriol. 1977;131:623–630. - PMC - PubMed
1. Ausubel, F. M., et al. (ed.). Current protocols in molecular biology. John Wiley & Sons, Inc., New York, N.Y.
1. Bajaj V, Hwang C, Lee C A. hilA is a novel ompR/toxR family member that activates the expression of Salmonella typhimurium invasion genes. Mol Microbiol. 1995;18:715–727. - PubMed
1. Bajaj V, Lucas R L, Hwang C, Lee C A. Co-ordinate regulation of Salmonella typhimurium invasion genes by environmental and regulatory factors is mediated by control of hilA expression. Mol Microbiol. 1996;22:703–714. - PubMed

OmpR regulates the two-component system SsrA-ssrB in Salmonella pathogenicity island 2 - PubMed (original) (raw)