Programmed Delay of a Virulence Circuit Promotes Salmonella Pathogenicity

To accomplish successful infection, pathogens must operate their virulence programs in a precise, time-sensitive, and coordinated manner. A major question is how pathogens control the timing of virulence gene expression during infection. Here we report that the intracellular pathogen Salmonella controls the timing and level of virulence gene expression by using an inhibitory protein, EIIANtr. A DNA binding master virulence regulator, PhoP, controls various virulence genes inside acidic phagosomes. Salmonella decreases EIIANtr amounts at acidic pH in a Lon- and PhoP-dependent manner. This, in turn, promotes expression of the PhoP-activated virulence program because EIIANtr hampers activation of PhoP-regulated genes by interfering with PhoP binding to DNA. EIIANtr enables Salmonella to impede the activation of PhoP-regulated gene expression inside macrophages. Our findings suggest that Salmonella achieves programmed delay of virulence gene activation by adjusting levels of an inhibitory factor.

an inhibitory factor shapes the activation of the master virulence regulatory twocomponent system in the intracellular pathogen Salmonella enterica.
Given that intracellular pathogens experience an acidic pH inside the host phagosome (2)(3)(4), it is important for them to have a system that can respond to pH changes in order to survive and cause disease inside the host (5)(6)(7)(8)(9). The Salmonella PhoP/PhoQ two-component system is a master virulence regulatory complex (10,11) that is activated by acidic pH (12,13), low Mg 2ϩ (14), and certain antimicrobial peptides (15). This system is crucial for Salmonella virulence because the lack of either PhoP or PhoQ impairs Salmonella pathogenicity (10,11). Activation of this system by acidic pH is critical for Salmonella virulence because the inhibition of acidification of the Salmonellacontaining vacuole prevents expression of PhoP-activated genes in phagocytic (9,16) and nonphagocytic (17) cells, limits replication inside macrophages (6,18), and attenuates virulence in mice (19). Although "turn on" of the PhoP/PhoQ system is necessary for virulence, it is also important to precisely control this system because constant activation of this system renders Salmonella avirulent in mice (20).
The ptsN gene encodes EIIA Ntr , a component of the nitrogen-metabolic phosphotransferase system (PTS) (21,22). This nitrogen-metabolic PTS lacks a membrane-bound complex that controls the activities of sugar PTSs in response to particular sugar availabilities (21,22). Recent studies have reported that EIIA Ntr is involved in various cellular functions, including potassium uptake (23,24), the stringent response (25,26), and amino sugar homeostasis (27). Moreover, we recently reported that EIIA Ntr promotes virulence by hampering SsrB, a transcriptional regulator of Salmonella pathogenicity island 2 (SPI-2) (28). Despite the fact that various regulatory functions of EIIA Ntr have been identified, the regulation of its own expression remains largely unknown.
Here we establish that Salmonella alters EIIA Ntr abundance, thereby controlling activation of the PhoP/PhoQ system during infection. Under acidic conditions, Salmonella reduces EIIA Ntr amounts by Lon-mediated degradation in a PhoP-dependent manner. EIIA Ntr hampers PhoP binding to its target DNA, thereby decreasing expression of PhoP-activated genes under acidic pH conditions. This double-negative regulation results in an overall positive feedback that furthers activation of the system. Our findings suggest that Salmonella ensures the timing and extent of its PhoP/PhoQmediated virulence program via regulation of an inhibitory factor during infection.

RESULTS
EIIA Ntr amounts decrease upon environmental acidification. To investigate the expression of EIIA Ntr , we first investigated its transcription levels by measuring ␤-galactosidase activity produced by a p rpoN -lacZ fusion given that the ptsN gene is located downstream of the rpoN gene, forming an operon (21). Although EIIA Ntr is a component of a nitrogen-metabolic PTS (21,22), transcription levels of rpoN remained unaltered by 100-fold changes in the concentration of a nitrogen source (Fig. 1A). We next examined rpoN expression at different pH values or concentrations of Mg 2ϩ , representing environmental conditions that Salmonella might encounter during infection (6,14). However, none of those changes modified the expression of rpoN ( Fig. 1B  and C).
Despite the absence of notable changes in p rpoN -lacZ expression, we examined EIIA Ntr protein amounts under these conditions. Similar to rpoN expression, EIIA Ntr amounts were not responsive to changes in nitrogen source (Fig. 1D). Surprisingly, however, EIIA Ntr abundance was significantly reduced when Salmonella was exposed to low-Mg 2ϩ or acidic-pH conditions ( Fig. 1E and F).
This rpoN-independent alteration of EIIA Ntr levels ( Fig. 1A to F) raised the possibility that transcription of the ptsN gene might not just be from the rpoN promoter. Indeed, primer extension analysis indicated the presence of a transcriptional start site 67 nucleotides upstream of the EIIA Ntr start codon (Fig. 1G). Therefore, we investigated the expression of a p ptsN -lacZ transcriptional fusion in bacteria grown under the abovedescribed conditions. However, none of those conditions altered the expression of p ptsN -lacZ ( Fig. 1H to J). Taken together, these results suggest that Salmonella probably controls EIIA Ntr levels through posttranscriptional regulatory mechanisms.
PhoP decreases EIIA Ntr abundance posttranscriptionally. Given that an acidic pH and low Mg 2ϩ are signals activating the sensor PhoQ (12)(13)(14), we hypothesized that the PhoP/PhoQ system might be involved in altering EIIA Ntr amounts. To test this hypothesis, we examined the transcription and translation levels of EIIA Ntr in isogenic wild-type and the phoP mutant Salmonella strains. Consistent with the expression of the p ptsN -lacZ fusion gene from a plasmid (Fig. 1H to J), the chromosomal ptsN-lacZ fusion also showed similar ␤-galactosidase activities under acidic and neutral pH conditions (see Fig. S1A in the supplemental material). Moreover, mutation of the phoP gene did not alter ptsN expression (Fig. S1A). In contrast, EIIA Ntr amounts were significantly higher in the phoP null mutant than in the wild type when grown under acidic conditions ( Fig. 2A), indicating that PhoP reduces EIIA Ntr amounts independent of its transcription. The increased EIIA Ntr abundance in the phoP mutant was restored by a plasmid expressing PhoP from a heterologous promoter ( Fig. 2A). The absence of the cognate sensor kinase PhoQ also coordinated with a higher abundance of EIIA Ntr (Fig. S1B) as in the phoP mutant ( Fig. 2A), indicating that PhoP's action in altering EIIA Ntr abundance is dependent on PhoP's phosphorylation. Furthermore, the lack of PhoP increased EIIA Ntr FIG 1 Acidic pH and low Mg 2ϩ conditions decrease EIIA Ntr levels. (A to C and H to J) ␤-Galactosidase activities were determined from wild-type Salmonella harboring a plasmid with a p rpoN -lacZ fusion (A to C) or a plasmid with the p ptsN -lacZ fusion (H to J). Bacteria were grown in M9 medium with the indicated variations. The means and SDs from three independent experiments are shown. (D to F) Western blot analysis of crude extracts prepared from Salmonella expressing EIIA Ntr -FLAG from its normal chromosomal location. Bacteria were grown in M9 medium with the indicated modifications (N, 2, 20, and 200 mM NH 4 Cl; Mg 2ϩ , 0.01 and 2 mM MgCl 2 ; pH, 5.8 and 7.0). Representative results from at least three independent experiments are shown. (G) Primer extension analysis of the ptsN gene using total RNA prepared from wild-type Salmonella grown in M9 medium. The bold "G" indicates the transcriptional start site (ϩ1) of the ptsN gene, located 67 nucleotides upstream from the start codon of the ptsN gene. Representative results from at least three independent experiments are shown. A schematic of the ptsN promoter with the rpoN gene is displayed on the right side.
Programmed Delay of Salmonella Virulence ® abundance ( Fig. 2B) even when transcription of ptsN was induced by isopropyl-␤-Dthiogalactopyranoside (IPTG), further supporting the notion that PhoP modulates the abundance of EIIA Ntr posttranscriptionally.
EIIA Ntr protein is degraded under acidic conditions in a PhoP-dependent manner. We next investigated how PhoP controls EIIA Ntr levels posttranscriptionally. PhoP may decrease EIIA Ntr levels by reducing the stabilities of ptsN mRNA and/or EIIA Ntr protein. ptsN mRNA showed similar levels of decay in both the wild type and the isogenic phoP mutant upon addition of rifampin to stop transcription (Fig. 2C). In contrast, the amount of EIIA Ntr decreased in the wild type after inhibition of protein synthesis with chloramphenicol treatment (half-life [t 1/2 ] Ͻ 60 min), whereas it remained constant in the strain lacking PhoP (t 1/2 Ͼ 120 min) (Fig. 2D). Furthermore, this EIIA Ntr degradation was detected when Salmonella was grown at an acidic pH but not at a neutral pH (Fig. S2). These results suggest that PhoP boosts the degradation of EIIA Ntr protein in an acidic environment.
Lon protease mediates PhoP-dependent degradation of EIIA Ntr . Cytoplasmic proteases, including ClpXP and Lon, are involved in the proteolysis of cytosolic proteins in Gram-negative bacteria (29), and EIIA Ntr is a cytoplasmic protein. As PhoP counteracts ClpXP-mediated proteolysis of RpoS via IraP (30), we first investigated the potential role of ClpXP in controlling EIIA Ntr abundance. A Salmonella strain lacking ClpXP produced amounts of EIIA Ntr comparable to those of the wild type, unlike the phoP mutant (Fig. 2E). However, a lack of Lon increased the abundance of EIIA Ntr protein compared with that of the wild type, similar to the case with the phoP mutant strain (Fig. 2E). If Lon is responsible for the degradation of EIIA Ntr , the lon mutant should make EIIA Ntr stable. Like the phoP mutant (Fig. 2D), the lon mutant displayed sustained abundance of EIIA Ntr after chloramphenicol treatment (t 1/2 Ͼ 120 min), whereas EIIA Ntr levels dwindled in the wild type (t 1/2 Ͻ 60 min) (Fig. 2F). Furthermore, double deletion of the phoP and lon genes resulted in amounts of EIIA Ntr comparable to those in the phoP or lon single-deletion mutants (Fig. S3). These results suggest that PhoP favors Lon protease-mediated degradation of EIIA Ntr .
EIIA Ntr negatively controls expression of PhoP-regulated genes. We next wondered why Salmonella curtails EIIA Ntr amounts when it encounters an acidic pH, a PhoP-inducing condition inside macrophage phagosomes (31). To understand the role of EIIA Ntr , we investigated genes that are regulated by EIIA Ntr using a DNA microarray experiments with wild-type and isogenic ptsN mutant strains grown in acidified minimal medium. We found 768 differentially expressed genes in the ptsN mutant compared with the wild type (Ͼ2-fold): 371 upregulated genes and 397 downregulated genes (Fig. S4). Consistent with a previous report (28), SPI-2 genes were more highly expressed in the ptsN mutant than in the wild type (Table S1). Interestingly, we found that transcript levels of PhoP-regulated genes were higher in the strain lacking EIIA Ntr than in the wild type (Table S1). We further verified the EIIA Ntr 's regulatory effects on PhoP-activated genes using quantitative reverse transcription-PCR (qRT-PCR): the ptsN mutant displayed 3-to ϳ7-fold-higher transcript levels of PhoP-regulated genes than the wild type (Fig. 3A). Moreover, the elevated expression of those genes in the ptsN mutant was restored to wild-type levels by a plasmid expressing the ptsN gene from a heterologous promoter but not by the plasmid vector (Fig. 3A). Interestingly, plasmiddriven heterologous expression of an unphosphorylatable variant of EIIA Ntr (H73A) or a variant of EIIA Ntr mimicking the phosphorylated form (H73E) (25) was also able to rescue the expression of PhoP-regulated genes similarly to wild-type EIIA Ntr (Fig. 3A). These results suggest that EIIA Ntr modulates the expression of PhoP target genes independent of EIIA Ntr 's phosphorylation status and PhoP transcription.
Control of PhoP-regulated genes by EIIA Ntr requires PhoP. We next wondered whether EIIA Ntr controls expression of PhoP to regulate PhoP regulon. If EIIA Ntr directly controls PhoP-activated genes, EIIA Ntr should be able to regulate those genes in the absence of PhoP. However, the absence of PhoP abrogated the regulatory effects of EIIA Ntr on the expression of PhoP-activated genes (Fig. 3B), indicating that control of the PhoP regulon by EIIA Ntr requires PhoP. If the regulation of PhoP-regulated genes by EIIA Ntr is due to altered phoP transcription, heterologous expression of phoP from a Programmed Delay of Salmonella Virulence ® plasmid should abolish the effect of EIIA Ntr on the expression of those genes. A lack of EIIA Ntr increased expression levels of PhoP-regulated genes, even when PhoP was produced from a heterologous promoter (Fig. 3C). These results indicate that EIIA Ntr regulates PhoP regulon in a PhoP-dependent manner.
EIIA Ntr hampers PhoP binding to its target promoter DNA. Given that EIIA Ntr regulates other regulatory systems via protein-protein interaction (23,24,28,32), we next investigated whether EIIA Ntr interacts with the PhoP protein. We used the bacterial two-hybrid system, in which ␤-galactosidase levels are dependent on the proximity of fused proteins to fragments (i.e., T25 and T18) of the Bordetella pertussis adenylate cyclase in an Escherichia coli strain lacking its own adenylate cyclase (33). Coexpression of T25-EIIA Ntr and T18-PhoP resulted in approximately 141-fold-higher levels of ␤-galactosidase activity than in strains expressing T25-EIIA Ntr and T18 fragment or empty vectors (Fig. 4A), indicating that EIIA Ntr interacts with PhoP. This activity was comparable to that from the positive-control strain harboring T25 and T18 fragments fused to the leucine zipper of the transcription factor GCN4 (Fig. 4A). Consistent with the observation that unphosphorylatable EIIA Ntr (H73A) functions like the wild type in controlling the PhoP regulon (Fig. 3A), EIIA Ntr (H73A) displayed an interaction with PhoP similar to that of the wild-type protein (Fig. 4A).
PhoP promotes transcription of the PhoP regulon by binding to DNA (34) when PhoP is activated by PhoQ-mediated phosphorylation under inducing conditions (35) or by reducing acetylation of PhoP (36). Thus, the interaction of EIIA Ntr with PhoP could decrease PhoP activity by inhibiting the interaction of PhoP with the cognate kinase PhoQ (i.e., reducing phosphorylation), by promoting acetylation of PhoP, or by reducing deacetylation of PhoP. Alternatively, EIIA Ntr could interfere with PhoP binding to DNA.
If EIIA Ntr hampers PhoP phosphorylation by PhoQ, the lack of PhoQ should abolish the regulatory effects of EIIA Ntr on the PhoP regulon. Because PhoP is not active in the absence of PhoQ, we investigated the function of EIIA Ntr in a phoP* phoQ strain lacking PhoQ and expressing a PhoP variant that autophosphorylates from acetyl phosphate (35). EIIA Ntr reduced pagD expression even in the absence of PhoQ (Fig. S5), indicating that the regulatory action of EIIA Ntr is independent of PhoQ. We next examined whether acetylation of PhoP is responsible for EIIA Ntr -mediated regulation of PhoP target genes by mutating known acetylase (Pat) or deacetylase (CobB) (36). However, EIIA Ntr displayed similar regulatory effects on pagD expression in the absence of Pat or CobB (Fig. S5).
To test whether EIIA Ntr inhibits PhoP's DNA binding ability, a gel shift assay was conducted using purified PhoP and EIIA Ntr proteins with the phoP-activated pagD promoter. Purified PhoP bound to the pagD promoter DNA and formed a complex with the probe DNA in vitro (Fig. 4B). EIIA Ntr prevented PhoP from binding to the target DNA: the PhoP-DNA complex decreased to generate the unbound pagD promoter DNA when amounts of EIIA Ntr increased (although an excess of EIIA Ntr could not fully dissociate PhoP from DNA), and EIIA Ntr alone did not form a complex with the DNA (Fig. 4B). However, addition of an EIIA Ntr paralogue, EIIA Glc , did not alter binding of PhoP to DNA, indicating that it is specific to EIIA Ntr (Fig. S6A). This inhibitory function of EIIA Ntr is specific, as another regulatory protein, PmrA, bound to the pbgP promoter DNA regardless of EIIA Ntr (Fig. S6B). Taken together, these data suggest that EIIA Ntr reduces expression of the PhoP regulon by inhibiting PhoP binding to its target promoter DNA.
EIIA Ntr delays activation of PhoP target genes inside macrophages. We next examined if EIIA Ntr could control the expression of PhoP-activated genes during infection. To evaluate this, macrophages were infected with wild-type and mutant Salmonella strains harboring a gfp fusion with the promoter of the PhoP-activated gene pagD, and fluorescence was measured. Activation of pagD expression inside macrophages was completely dependent on PhoP, because the phoP mutant was unable to produce any fluorescence, in contrast to the wild type (Fig. 5A). The ptsN mutant showed higher and earlier activation of pagD expression than the wild type (Fig. 5A), indicating that EIIA Ntr inhibits PhoP activation inside macrophages. These results suggest that Salmonella delays expression of PhoP-activated genes inside macrophages via EIIA Ntr . The ptsN mutant also displayed accelerated activation of the pagD gene compared to that of the wild type in acidic pH (Fig. S7A). Consistent with a previous report (28), the lack of EIIA Ntr rendered Salmonella virulence attenuated in mice inoculated via the intraperitoneal route (Fig. 5B, left). Defective virulence of the ptsN mutant Salmonella was also observed when mice were inoculated via the oral route (Fig. 5B, right). These results are in agreement that EIIA Ntr controls the expression of Programmed Delay of Salmonella Virulence ® various virulence genes, including the PhoP and SsrB regulons (Fig. 3) (28). And it is possible that the delayed virulence gene expression by EIIA Ntr might be critical for Salmonella pathogenicity.

DISCUSSION
In this study, we established that Salmonella employs EIIA Ntr to delay the activation of its virulence program inside acidic phagosomes (Fig. 6). The master virulence regulator PhoP promotes the Lon-mediated degradation of EIIA Ntr under acidic conditions ( Fig. 1 and 2); this, in turn, favors the expression of PhoP-activated genes under acidic conditions (Fig. 3) and inside macrophages (Fig. 5A). Thus, the reduction of EIIA Ntr amounts in the acidic phagosome allows delayed but robust activation of the Salmonella virulence program, including the PhoP/PhoQ system as well as SPI-2 genes (28) (Fig. 6), thereby enhancing its fitness inside the host (11, 28) (Fig. 5B).
Although nitrogen availability controls the phosphorylation status of EIIA Ntr (37), total amounts of EIIA Ntr remain unaltered in vivo (37) (Fig. 1D), suggesting that EIIA Ntr phosphorylation status is important for its regulatory function. However, the role of EIIA Ntr phosphorylation in its regulatory function is controversial: some EIIA Ntr activities are dependent on its phosphorylation status (23,24,26,27), whereas others are not (24,25,27,28,32,38). EIIA Ntr interacts with PhoP and decreases expression of PhoPactivated genes regardless of its phosphorylation status ( Fig. 3A and Fig. 4A). Moreover, EIIA Ntr accumulated in the phoP mutant of even in the absence of EI Ntr (encoded by the ptsP gene), the phosphor donor for EIIA Ntr (Fig. S8). Together with previous reports (24,25,27,28,32,38), these findings indicate that EIIA Ntr operates some functions regardless of its phosphorylation status, suggesting that it is important to understand how bacteria control cellular amounts of EIIA Ntr protein.
In this study, we established that Salmonella modulates EIIA Ntr abundance via Lon-mediated degradation in a PhoP-dependent manner (Fig. 2). Moreover, we demonstrated that the ptsN gene has its own transcriptional start site, although the ptsN gene is considered a component of the rpoN operon (21). This raises the possibility that Salmonella may control expression of ptsN independent of rpoN. Indeed, a recent transcriptome sequencing (RNA-seq) study showed that nitrogen oxide shock reduces ptsN transcript levels but does not alter rpoN mRNA levels (39). Moreover, a recent study has shown that EIIA Ntr is degraded by Lon in the absence of GlmS and N-acetylglucosamine, although this degradation was not observed in the wild type (27). Furthermore, EIIA Ntr accumulates in the presence of acetylglucosamine in a degradation-independent manner (27). As a transcriptional regulator, PhoP probably induces an adapter-like protein that can alter degradability of EIIA Ntr by Lon in a PhoP-dependent manner; rather, PhoP brings EIIA Ntr to Lon. PhoP controls 9% of Salmonella genes (40) despite the fact that limited numbers of its direct targets are known. Given that EIIA Ntr regulates 768 genes (Fig. S4), PhoP perhaps controls a subset of genes via EIIA Ntr .
Our findings now provide mechanisms for how EIIA Ntr contributes to Salmonella virulence. EIIA Ntr tunes the timing and extent of virulence regulatory systems' activation inside host cells (Fig. 3A and Fig. 5A) (28), thereby enabling Salmonella to properly manage its virulence program. In addition, there are other biological processes regulated by EIIA Ntr , and they are potentially involved in bacterial virulence, which includes ppGpp accumulation, metabolism to produce amino sugars, and potassium uptake (23,24,26,27). Thus, Salmonella probably changes various processes by altering EIIA Ntr abundance during infection.
The function of EIIA Ntr described here may explain the different behaviors of PhoP-regulated genes in in vitro cultures and inside macrophages; the expression of PhoP-activated genes reaches maximal levels 5 to ϳ25 min after exposure to an environment that activates PhoQ (41), whereas it takes hours inside macrophages (16,42) (Fig. 5A). Moreover, full activation of PhoP in acidic pH requires a PhoP-activated UgtL protein which amplifies the response of PhoQ to an acidic environment (43). Because EIIA Ntr binds to and hampers SsrB (28), induction of SsrB would favor PhoP activation by reducing the number of EIIA Ntr proteins interacting with PhoP. Moreover, inhibition of PhoP enables EIIA Ntr to efficiently hamper the SsrB regulon given that PhoP transcriptionally activates SsrB (44) and that EIIA Ntr hinders SsrB's regulatory function (28).
Although the activation of the pagD gene inside macrophages was delayed in the wild type, maximal levels of activation were similar in the wild type and the ptsN mutant (Fig. 5A). In acidified defined media, however, the wild-type and the ptsN mutant strains did not show similar maximal levels of pagD expression (Fig. 3 and Fig. S7A). This might be due to difference in conditions that Salmonella experiences: acidic pH in defined media and complicated conditions inside acidic phagosomes. Moreover, PhoQ responds to multiple signals, including acidic pH, low Mg 2ϩ , antimicrobial peptides, and high osmolarity (45). EIIA Ntr reduces pagD gene expression not only at acidic pH ( Fig. 3 and Fig. S5) but also under conditions stimulating PhoQ, low Mg 2ϩ (Fig. S7B), and antimicrobial peptide C18G (Fig. S7C). Thus, we want to note that other PhoQ-inducing signals and/or other components inside phagosomes potentially contributing to the activation of the PhoP/PhoQ system inside phagosomes probably play a role in modulating EIIA Ntr -mediated function during infection.
Pathogens possess virulence genes that enable them to cause disease in the host. The EIIA Ntr gene can be defined as a virulence gene because it promotes Salmonella virulence in mice (28) (Fig. 5B). Paradoxically, EIIA Ntr antagonizes the functions of other virulence regulatory systems, such as PhoP/PhoQ and SsrB/SpiR, although the deletion of them highly attenuates Salmonella pathogenicity (11,46). This inhibition of virulence regulators by EIIA Ntr delays the timing of their activation (Fig. 5A) and probably allows robust activation once the amount of active regulator(s) supersedes the inhibitory threshold created by EIIA Ntr (Fig. 6).
Why does Salmonella limit activation of virulence regulatory systems via EIIA Ntr during infection, although this may potentially decrease its pathogenicity? One possible explanation is that overactivation of those virulence systems might be harmful to Salmonella survival inside the host. Hyperconstitutive activation of the PhoP/PhoQ system actually attenuates Salmonella virulence in mice (20). Moreover, PhoP activates not only virulence factors but also antivirulence factors (47)(48)(49). Balancing those virulence and antivirulence factors is probably important to achieving optimal fitness inside the host. In addition, it is possible that retarding induction of the PhoP-activated virulence program may allow Salmonella to efficiently replicate and spread to other cells. Because PhoP-activated SPI-2 genes result in macrophage death (50,51) and early activation of SPI-2 genes accelerates cell death (48), delayed activation of SPI-2 genes probably allows Salmonella sufficient time to replicate inside the host cell.
Furthermore, EIIA Ntr may also help Salmonella efficiently turn off those systems when unnecessary (e.g., when Salmonella escapes from phagocytes). Efficient transition between the "on" and "off" states of virulence regulatory systems allows the bacterial virulence program to operate efficiently and saves energy by reducing unnecessary usage.

MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. The Salmonella enterica serovar Typhimurium strains used in this study were derived from strain SL1344. The strains and plasmids used in this study are listed in Table S2A. Phage P22-mediated transduction was performed as described previously (52). All Salmonella strains were grown aerobically at 30 or 37°C in Luria-Bertani (LB) or M9 minimal medium at the desired pH and Mg 2ϩ concentrations to mid-to late log phase unless specified. Antimicrobial peptide C18G was treated at 5 g/ml for an hour. Antibiotics were used at the following concentrations: ampicillin, 50 g/ml; chloramphenicol, 25 g/ml; and kanamycin, 50 g/ml. Primers used for the construction of bacterial strains and plasmids are listed in Table S2B.
Construction of mutant Salmonella strains. To generate a ptsN-FLAG strain, a cat cassette was introduced in the 3= end of the ptsN gene as follows: the cat fragment was amplified from pKD3 using primers ptsN-FLAG-F/ptsN-FLAG-R and then introduced into wild-type Salmonella (SL1344) harboring plasmid pKD46 as previously described (53). The cat cassette was removed with plasmid pCP20 (53).
To generate a ptsP mutant, a cat fragment was amplified from pKD3 using primers ptsP-Red-F/ptsP-Red-R and then introduced into wild-type Salmonella harboring plasmid pKD46 (53).
To generate a phoP mutant strain, a kan fragment was amplified from pKD13 using primers phoP-Red-F/phoP-Red-R and then introduced into wild-type Salmonella harboring plasmid pKD46 (53). Next, the kan cassette was removed with plasmid pCP20 (53).
To generate a phoQ mutant strain, a kan fragment was amplified from pKD13 using primers phoQ-Red-F/phoQ-Red-R and then introduced into wild-type Salmonella harboring plasmid pKD46 (53). Next, the kan cassette was removed with plasmid pCP20 (53).
To generate a cobB mutant strain, a kan fragment was amplified from pKD13 using primers cobB-P1-F-kan/cobB-P4-R-kan and then introduced into wild-type Salmonella harboring plasmid pKD46 (53).
To generate a pat mutant strain, a kan fragment was amplified from pKD13 using primers pat-P1-F-kan/pat-P4-R-kan and then introduced into wild-type Salmonella harboring plasmid pKD46 (53).
To generate a clpXP mutant, a cat fragment was amplified from pKD3 using primers clpP-Red-F/ clpX-Red-R2 and then introduced into wild-type Salmonella harboring plasmid pKD46 (53). Next, the kan cassette was removed with plasmid pCP20 (53).
To generate a lon mutant, a cat fragment was amplified from pKD3 using primers lon-Red-F/lon-Red-R and then introduced into wild-type Salmonella harboring plasmid pKD46 (53). Next, the kan cassette was removed with plasmid pCP20 (53).
To generate a strain with the p ptsN -lacZ fusion in the normal chromosomal location, pCP20 was introduced into SR3203 (the ptsN mutant). Next, the lacZ fusion was generated with plasmid pCE70 (54).
To generate a strain with the p pagD -lacZ fusion in the normal chromosomal location, a cat fragment was amplified from pKD3 using primers pagD-1/pagD-2 and then introduced into wild-type Salmonella (SL1344) harboring plasmid pKD46 as previously described (53). The cat cassette was removed with plasmid pCP20 (53). Next, the lacZ fusion was generated with plasmid pCE70 (54).
A plasmid expressing the phoP gene was constructed as follows: the phoP coding region was amplified from wild-type Salmonella (SL1344) using primers phoP-com-F2/phoP-com-R2 and then introduced between the EcoRI and BamHI sites of pUHE21-2lacI q (55).
A plasmid expressing phoQ gene was constructed as follows: the phoQ coding region was amplified from wild-type Salmonella (SL1344) using primers phoQ-com-F/phoQ-com-R and then introduced between the EcoRI and BamHI sites of pUHE21-2lacI q (55).
A plasmid expressing His 6 -tagged PhoP was constructed as follows: the phoP coding region was amplified from wild-type Salmonella (SL1344) using primers pPhoP-F/pPhoP-His6-R and then introduced between the BamHI and HindIII sites of pUHE21-2lacI q (55).
A plasmid expressing His 6 -tagged PmrA was constructed as follows: the pmrA coding region was amplified from wild-type Salmonella (SL1344) using primers pPmrA-F/pPmrA-His6-R and then introduced between the BamHI and HindIII sites of pUHE21-2lacI q (55).
A plasmid expressing T18-PhoP fusion protein was constructed as follows: the phoP gene was amplified from wild-type Salmonella (SL1344) using primers phoP-F2/phoP-R1 and then introduced between the BamHI and EcoRI sites of pUT18C (58).
Western blotting. Salmonella strains expressing the EIIA Ntr -FLAG protein from its normal chromosomal location or under the control of a heterologous promoter were grown as described in "Bacterial strains, plasmids, and growth conditions," above. Bacteria were collected by centrifugation, and cell lysates were prepared using B-PER solution (Pierce). Cell lysates were separated by 12% SDS-PAGE, and EIIA Ntr and DnaK were detected using anti-FLAG (Sigma) and anti-DnaK (Abcam) antibodies, respectively. Blots were developed using anti-mouse IgG horseradish peroxidase-linked antibody with the ECL detection system (Amersham Biosciences).
␤-Galactosidase assay. ␤-Galactosidase assays were carried out in triplicate, and the activity was determined as described previously (59).
RNA isolation and quantitative RT (qRT)-PCR. Salmonella strains were grown as described above, and total RNA was isolated using an RNeasy minikit (Qiagen). After DNase treatment of the isolated RNA, cDNA was synthesized using Omnitranscript reverse transcription reagents (Qiagen) and random hexamers (Invitrogen). Quantification of the cDNA was carried out using 2ϫ iQ SYBR Green Supermix (Bio-Rad), and real-time amplification of the PCR products was performed using the iCycler iQ real-time detection system (Bio-Rad). The primers used for detection of the gene transcripts are listed in Table S2B. Data were normalized to the abundance of 16S rRNA expression levels.
RNA or protein stability analyses. To test RNA stability, bacterial cultures were treated with 0.1 mg/ml of rifampin to stop transcription, and samples were collected at the desired time points. Total RNA was isolated, and mRNA levels were determined by qRT-PCR as described above. To test the protein stability, bacterial cultures were treated with 0.2 mg/ml of chloramphenicol to stop protein synthesis, and samples were collected at the desired time points. Protein levels were analyzed using Western blot analysis.
Bacterial two-hybrid assay. E. coli BTH101 organisms harboring derivatives of plasmids pUT18 and pKT25 were grown overnight in LB broth containing ampicillin (100 g/ml) and kanamycin (50 g/ml), adding a 1:100 dilution to 1 ml of the same fresh medium containing 0.5 mM IPTG and employing shaking at 30°C overnight as previously described (33,58).
Purification of proteins. His 6 -tagged PhoP, His 6 -tagged PmrA and His 6 -tagged EIIA Ntr were expressed in E. coli BL21(DE3). Bacterial cells were grown in LB medium at 37°C until the optical density at 600 nm (OD 600 ) reached 0.5, and the expression of those proteins was induced by addition of IPTG (0.5 M) followed by growth at 30°C for 5 h. Cells were harvested, washed, and suspended in buffer A (20 mM Tris [pH 8.0], 150 mM NaCl, and 20 mM imidazole). Then the cells were disrupted by sonication, and cell debris was removed by centrifugation at 20,000 ϫ g at 4°C for 30 min. The supernatant was applied to a 1.5-ml nickel-nitrilotriacetic acid (Ni-NTA) agarose column equilibrated in buffer A, washed with a 25-column volume of the same buffer, and eluted using a gradient of buffer A and buffer B (20 mM Tris [pH 8.0], 150 mM NaCl, and 250 mM imidazole). The fractions were then collected and analyzed by SDS-PAGE, and selected fractions were dialyzed against buffer C (20 mM Tris [pH 8.0], 150 mM NaCl, and 10% glycerol).
EMSA. DNA fragments containing the promoter region of the pagD or pbgP gene were amplified by PCR using the primers EMSA-pagD-F/EMSA-pagD-R and EMSA-pbgP-F/EMSA-pbgP-R, respectively. Purified promoter DNA (80 fmol) was incubated with the desireded concentrations of purified PhoP-His 6 or PmrA-His 6 with EIIA Ntr -His 6 or EIIA Glc -His 6 at room temperature for 20 min in 15 l of binding buffer (10 mM Tris [pH 7.5], 0.5 mM EDTA, 1 mM MgCl 2 , 0.5 mM dithiothreitol [DTT], and 50 mM NaCl) containing 5 ng/l of poly(dI-dC). Samples were prepared by addition of 3 l of 6ϫ electrophoretic mobility shift assay (EMSA) gel loading solution and separated by electrophoresis using a 6% nondenaturing polyacrylamide gel. DNA staining was performed according to the manufacturer's instructions (EMSA kit; E33075; Thermo Fisher Scientific).
Macrophage infection assay. The murine-derived macrophage line RAW264.7 was cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies) supplemented with 10% heatinactivated fetal bovine serum (FBS; Life Technologies) at 37°C with 5% CO 2 . Macrophages were seeded in 24-well tissue culture plates at 5 ϫ 10 5 per well 1 day before infection with Salmonella. Confluent monolayers were inoculated with bacterial cells that had been grown overnight in LB broth, washed with phosphate-buffered saline (PBS), and resuspended in 0.1 ml of prewarmed DMEM at a multiplicity of infection of 20. Following a 30-min incubation, the wells were washed three times with prewarmed PBS to remove extracellular bacteria and then incubated with prewarmed medium supplemented with 100 g/ml of gentamicin for 1 h to kill extracellular bacteria. Next, the wells were washed three times with prewarmed PBS and incubated with prewarmed medium supplemented with 10 g/ml of gentamicin. At the desired time points, the cells were washed three times with prewarmed PBS and subjected to the following procedures. For green fluorescent protein (GFP) assessment, washed cells were scraped with 200 l of PBS and subjected to fluorescence measurements at 510 nm. For CFU measurements, washed cells were lysed with PBS containing 1% Triton X-100 and plated on LB agar plate at the proper dilutions.
Mouse virulence assay. Six-week-old female BALB/c mice were purchased from the Institute of Laboratory Animal Resources at Seoul National University. Five mice in each group were infected intraperitoneally or orally with 0.1 ml of PBS containing approximately 10 2 or 10 6 Salmonella cells grown in LB broth overnight, respectively. All animals were housed in temperature-and humidity-controlled rooms and maintained on a 12-h light/12-h dark cycle. All procedures complied with the regulations of the Institutional Animal Care and Use Committee of Seoul National University.
Transcriptomic analysis. RNA labeling, hybridization to the microarrays, scanning, and data analysis were performed at Macrogen. Triplicates of total RNAs from wild-type and ptsN mutant strains grown in acidified M9 medium (pH 5.8) were purified as described above and subjected to microarray using a CombiMatrix chip for the Salmonella Typhimurium SL1344 genome (12,396 probes covering 4,441 genes). Arrays were scanned using the Axon GenePix 4000B scanner (Molecular Devices LLC). Image analysis and feature extraction were performed using Axon GenePix Pro software (Molecular Devices). The data were analyzed using Avadis Prophetic software version 3.3 (Strand Genomics). Fold changes were calculated by comparing averaged normalized signal intensities in wild-type versus ptsN mutant Salmonella. The t test was performed in parallel with the use of a false-discovery rate correction for multiple testing (60). A P value of Ͻ0.05 was used to pinpoint significantly different expression levels of genes. A cutoff of a 2-fold change for up-or downregulated expression was chosen to define genes that were differentially expressed.
Mapping of the transcription start site (primer extension assay). Reverse transcription was conducted using ptsN-P1 and Superscript II (Invitrogen). The ladder was generated with a template DNA that was amplified using primers ptsN-PE-F/ptsN-PE-R and the genomic DNA of SL1344.