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A Positive Feedback Loop Promotes Transcription Surge That Jump-Starts Salmonella Virulence Circuit

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Science  08 Dec 2006:
Vol. 314, Issue 5805, pp. 1607-1609
DOI: 10.1126/science.1134930

Abstract

The PhoP/PhoQ two-component system is a master regulator of Salmonella pathogenicity. Here we report that induction of the PhoP/PhoQ system results in an initial surge of PhoP phosphorylation; the occupancy of target promoters by the PhoP protein; and the transcription of PhoP-activated genes, which then subsides to reach new steady-state levels. This surge in PhoP activity is due to PhoP positively activating its own transcription, because a strain constitutively expressing the PhoP protein attained steady-state levels of activation asymptotically, without the surge. The strain constitutively expressing the PhoP protein was attenuated for virulence in mice, demonstrating that the surge conferred by PhoP's positive feedback loop is necessary to jump-start Salmonella's virulence program.

Unlike obligate parasites, which live in relatively constant environments, free-living organisms need to modulate their gene expression patterns in response to environmental cues. This is conspicuously true for bacterial pathogens, which must express (or silence) distinct sets of genes in the various tissues invaded during infection. This ensures that the encoded products are produced in the correct locales, at the required amounts and for the appropriate extents of time. Consequently, a pathogen's ability to colonize a particular niche and to cause disease is often compromised not only when a virulence regulatory factor is removed but also when it is constitutively activated (1, 2).

The PhoP/PhoQ two-component system is a major regulator of virulence in several Gram-negative species (3). Inactivation of the phoP or phoQ genes renders Salmonella enterica serovar Typhimurium five orders of magnitude less virulent for mice and unable to proliferate within phagocytic cells (46). The regulatory protein PhoP governs expression of ∼3% of the Salmonella genome (7) in response to the Mg2+ levels sensed by the PhoQ protein: Transcription of PhoP-activated genes is induced in low Mg2+ concentrations and repressed in high Mg2+ concentrations (8), whereas the converse is true for PhoP-repressed determinants. In addition to low Mg2+ concentrations, certain antimicrobial peptides have been shown to promote expression of some PhoP-activated genes at intermediate Mg2+ concentrations (9, 10).

Previously, the expression of PhoP-regulated genes was examined hours after activation, often by means of stable reporters such as β-galactosidase. To determine the changes in gene transcription taking place immediately after activation of the PhoP/PhoQ system, we investigated the expression kinetics of PhoP-regulated genes by isolating RNA as early as 5 min after Salmonella were shifted from media containing repressing (10 mM) to activating (50 μM) Mg2+ concentrations. The mRNA levels of the PhoP-activated mgtA, phoP, pmrD, and mig-14 genes increased after the shift to low Mg2+ concentration, reached a peak, and then decreased to attain steady-state levels that were 20 to 50% of the maximum (Fig. 1, A to D). A similar kinetic behavior was observed when Salmonella were induced in media with 200 μM Mg2+, which activates the PhoP/PhoQ system less than does 50 μMMg2+ (8): The mRNA levels of the PhoP-activated genes increased, reached a peak that was lower than that obtained when Salmonella was induced at 50 μMMg2+ (except for the mgtA gene, which reached the same levels), and then decreased (Fig. 1, A to D). These results demonstrated that activation of the PhoP/PhoQ system promotes a surge in the mRNA levels of PhoP-regulated genes, and that this surge is not specific to a particular PhoP-activated gene or inducing condition (11).

Fig. 1.

Induction of the PhoP/PhoQ system by the low-Mg2+ signal results in a surge in PhoP-regulated gene transcription. (A to D) The mRNA levels of the mgtA, phoP, pmrD, and mig-14 genes determined by real-time polymerase chain reaction (PCR) analysis using RNA prepared from wild-type (EG13918) (10) cells that were grown in medium containing 10 mM Mg2+, shifted to medium with either 50 μM(redlines) or 200 μM (blue lines) Mg2+, and harvested at the designated times. Expression levels were normalized to those of the 16S ribosomal RNA gene.

To examine whether the observed changes in mRNA levels were due to binding of the PhoP protein to its target promoters, we carried out chromatin immunoprecipitation (ChIP) experiments in organisms that were shifted from media with repressing (10 mM) to inducing (50 μM) Mg2+ concentrations. Occupancy of the mgtA and pmrD promoters by the PhoP protein, which was detected immediately after the shift, peaked at 20 min and then decreased to reach new steady-state levels (Fig. 2A) (12).

Fig. 2.

The surge in promoter occupancy by the PhoP protein is due to changes in the levels of phosphorylated PhoP protein. (A) Promoter occupancy by the PhoP protein determined by ChIP assay using wild-type (EG13918) cells that were grown in repressing (10 mM) Mg2+ concentrations, shifted to activating (50 μM) Mg2+ concentrations, and harvested after the addition of formaldehyde at the indicated times. Occupancy was quantified using real-time PCR analysis. The values of PhoP binding were obtained from normalization of PhoP occupancy (ratio of DNA bound by the PhoP protein to DNA not bound by the PhoP protein) of the target promoter to that of the endogenous control rpoD promoter. Error bars show the standard deviation from the mean. The levels of total (B) and phosphorylated (C) PhoP protein were determined using extracts from EG13918 cells that were grown in medium containing a high (10 mM) Mg2+ concentration, shifted to medium with low (50 μM) Mg2+ concentration, and harvested at the designated times. The levels of phospho-PhoP (P-PhoP) were quantified by phosphoimager analysis and normalized to the maximal level. The extracts were prepared from aliquots of cells normalized by optical density at 600 mm. and used for both Western blot and immunoprecipitation experiments.

The decrease in promoter occupancy and gene transcription taking place 20 min after induction of the PhoP/PhoQ system could reflect degradation of the PhoP protein and/or a reduction of its activity. We could rule out the first possibility because the PhoP protein levels increased for at least 60 min (Fig. 2B), which is in agreement with the PhoP/PhoQ system autogenously activating its own expression (Fig. 1B) (13). We have previously demonstrated that phosphorylated PhoP is the form of the PhoP protein that binds to its target promoters and activates gene transcription in vivo (14). Thus, we investigated the levels of phosphorylated PhoP protein in organisms that were grown under repressing (10 mM) Mg2+ concentrations and were then shifted to media with inducing (50 μM) Mg2+ concentrations and pulsed with 32PO4. Immunoprecipitation of the PhoP protein revealed that the levels of phospho-PhoP increased after the shift to low-Mg2+ media, peaking at 15 min; were decreasing by 30 min; and stabilized at 20% of the maximum levels by 60 min (Fig. 2C). This is in spite of the fact that the total amount of PhoP protein increased during this time frame (Fig. 2B). Together, these data suggest that the changes in promoter occupancy by the PhoP protein and in mRNA amounts of PhoP-activated genes reflect the levels of phospho-PhoP protein.

The PhoP and PhoQ proteins are encoded in a bi-cistronic operon that is transcribed from two promoters: a constitutive promoter that provides the basal levels of these proteins required for sensing and responding to changes in environmental conditions, and a regulated promoter that is activated by the PhoP protein when the bacterium experiences the low-Mg2+ inducing signal (8, 13). To test the possibility that positive autoregulation of the phoPQ operon is involved in the peak of activity of the phospho-PhoP protein, we compared the kinetics of promoter occupancy and gene transcription in two isogenic strains: one with the wild-type phoPQ promoter [that is, harboring the PhoP box that is responsible for transcriptional autoregulation (13)] and one in which the PhoP box was replaced by a consensus –35 sequence (Fig. 3A). Western blot analysis of extracts prepared from the strain with the wild-type phoPQ promoter revealed that the PhoP protein was detected only in organisms induced in low Mg2+ concentrations, which promoted a continuous increase in the PhoP protein levels during the first 45 min (Fig. 3B). In contrast, the strain with the –35 sequence in the phoPQ promoter produced the PhoP protein constitutively (Fig. 3B) at levels that were similar to the steady-state levels achieved by the strain with the wild-type phoPQ promoter after induction of the PhoP/PhoQ system (Fig. 3B). This allowed us to compare promoter occupancy and PhoP-mediated transcription in strains that differed only in the time required to produce steady-state levels of the PhoP protein. Despite synthesizing the PhoP protein constitutively, there was no occupancy of PhoP-activated promoters (Fig. 3C) or transcription of PhoP-activated genes (Fig. 3D) in the strain with the –35 sequence in the phoPQ promoter growing under repressing conditions.

Fig. 3.

The positive feedback loop of the phoPQ operon is necessary for the surge in activity of the PhoP/PhoQ system. (A) Schematic representation of the phoPQ promoter in isogenic strains EG13918 (top) and EG14943 (bottom). Strain EG13918 harbors the wild-type P1 promoter, which is positively autoregulated by the PhoP protein and the constitutive P2 promoter (8, 13). Strain EG14943 harbors a consensus –35 hexameric sequence (red square) in place of the PhoP box (blue square). The black square indicates the “scar” sequence generated during the construction of the strains (34). (B) The levels of total PhoP protein were determined using extracts prepared from equivalent numbers of EG13918 (top) and EG14943 (bottom) cells after switching Mg2+ concentrations as described in Fig. 2. The levels of promoter occupancy by the PhoP protein (C) and mRNA expression (D) of the mgtA and pmrD genes were determined in strains EG13918 (blue) and EG14943 (red) that were shifted from media with 10 mM to 50 μMMg2+ concentrations. The values for PhoP binding and mRNA expression were obtained as described in the legends of Figs. 1 and 2.

When the strain expressing the PhoP protein constitutively was shifted from repressing (10 mM) to inducing (50 μM) Mg2+ concentrations, binding of PhoP to the mgtA and pmrD promoters (Fig. 3C) and transcription of the respective genes (Fig. 3D) increased during the first 10 min and then asymptotically reached the steady-state levels displayed by the strain with the wild-type phoPQ promoter (15). In agreement with these results, the levels of phospho-PhoP increased upon the shift from high to low Mg2+ concentration and reached constant levels that were only ∼20% of the peak levels exhibited by the strain with the wild-type phoPQ promoter. Thus, positive autoregulation of the PhoP/PhoQ system is necessary for the surge in activity triggered by the low-Mg2+ inducing signal.

To determine whether the autoregulation-dependent surge in PhoP activity has a role in virulence, mice were inoculated with the two isogenic strains differing in the phoPQ promoter. All the mice inoculated with Salmonella bearing the wild-type phoPQ promoter died. In contrast, all the mice inoculated with the strain harboring the mutant phoPQ promoter with the –35 sequence replacing the PhoP box and expressing the PhoP protein constitutively survived (Fig. 4) (16). These results demonstrate that the surge in phospho-PhoP activity conferred by the PhoP/PhoQ positive feedback loop is essential for Salmonella virulence. Moreover, they imply that Salmonella's ability to cause a lethal infection in mice requires the rapid expression of PhoP-activated gene products and/or rapid repression of PhoP-repressed gene products. These might include virulence regulatory proteins such as the two-component system SpiR/SsrB (17), the transcriptional activators SlyA (18) and HilA (19), the alternative sigma factor RpoS (20), and/or virulence structural proteins such as MgtC (21), Mig-14 (22), and NagA (23).

Fig. 4.

Positive autoregulation of the phoPQ operon is required for Salmonella virulence in mice. C3H/HeN mice were injected intraperitoneally with ∼103 cells of wild-type Salmonella (EG13918, blue) and its isogenic mutant constitutively expressing the PhoP protein (EG14943 strain, red). None of the mice infected with EG14943 strain died during the first 3 weeks, whereas all the mice infected with strain EG13918 with the wild-type phoPQ promoter died. The survival assay was performed twice independently with groups of five mice per strain. Shown is the result of one of the two experiments, which gave similar results. The survival kinetics of mice infected with wild-type Salmonella strain 14028s was similar to that of mice infected with strain EG13918 (not shown in the figure).

The PhoQ protein can modify the levels of phospho-PhoP in vitro (24, 25), suggesting that it was probably responsible for the changes in the levels of phospho-PhoP observed in vivo (Fig. 2C). Thus, we compared the behavior of two isogenic strains expressing either a wild-type PhoQ protein or one with a single amino acid substitution that compromises its phosphatase activity (14). We used strains in which the chromosomal copy of the phoPQ operon had been deleted and that harbored a plasmid with the phoP-HA and phoQ genes under the control of a derivative of the lac promoter (14) because a strain with a chromosomal phoQ mutation encoding a phosphatase-defective PhoQ would constitutively activate the PhoP protein. We were able to identify a condition (0.5 mM isopropyl-β-d-thiogalactopyranoside and 33 μMMg2+) for the strain expressing the wild-type PhoQ protein that promoted a transcription pattern (fig. S3A) mimicking that displayed by the autoregulated strain induced in low-Mg2+ media (Fig. 1), reflecting the levels of phospho-PhoP protein in the cell (fig. S3B). In contrast, the mRNA levels of PhoP-activated genes did not decrease after the initial increase in the isogenic strain expressing the mutant PhoQ protein (fig. S3A), which is consistent with the persistently high levels of phospho-PhoP protein (fig. S3C). These results demonstrate that the PhoQ protein governs the levels of phospho-PhoP protein in vivo. Furthermore, they suggest that there is a temporal change in the kinase and phosphatase activities of PhoQ after activation of the PhoP/PhoQ system.

The activation surge displayed by PhoP/PhoQ is exhibited by other two-component systems. For example, activation of the PmrA/PmrB system (26, 27) by growing Salmonella in media containing a high (10 mM) Mg2+ concentration and then shifting it to media containing a low (50 μM) Mg2+ concentration and 100 μMFe3+ resulted in an increase in the mRNA levels corresponding to the PmrA-activated pbgP and pmrC genes, which peaked and then reached new steady-state levels (fig. S4A), reflecting changes in the amount of phospho-PmrA protein (fig. S4B). These data indicate that, like the PhoP/PhoQ system, the positively autoregulated PmrA/PmrB system (28, 29) responds to its specific signal by promoting an initial activation followed by lower steady-state levels.

A transient increase in the mRNA levels of the targets of regulation of the copper-responding CusR/CusS system from Escherichia coli (30), the vancomycin-responding VanR/VanS system from Streptomyces coelicolor (31), and the peptide pheromone–responding ComE/ComD system from Streptococcus pneumoniae (32) has also been observed when these two-component systems were activated by their respective signals. Additionally, constitutive expression of the comDE genes inhibited the development of competence in S. pneumoniae (33). Cumulatively, these findings indicate that the activation surge described for PhoP/PhoQ (Figs. 1 and 2) and PmrA/PmrB (fig. S4) is not exclusive to virulence-related systems from Salmonella and may be exhibited by systems having different physiological functions in diverse bacterial species. The one correlating factor is that all these systems positively autoregulate their own expression, which in the case of the Salmonella PhoP/PhoQ system is required for the normal surge of activity (Fig. 2) and virulence (Fig. 4) in mice.

What is the significance of response curves (Figs. 1 and 2 and fig. S4) in which regulatory systems display a peak of activity before reaching new steady-state levels? The initial activation may allow the immediate establishment of a new phenotypic state. This would enable an organism to carry out the necessary tasks required to face the condition that triggered activation of the regulatory system. The steady-state levels of expression that follow the initial activation would then serve to maintain the new phenotypic state.

Supporting Online Material

www.sciencemag.org/cgi/content/full/314/5805/1607/DC1

Materials and Methods

Figs. S1 to S5

Tables S1 to S3

References

References and Notes

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