The roles of SsrA–SsrB and OmpR–EnvZ in the regulation of genes encoding the Salmonella typhimurium SPI-2 type III secretion system

Junkal Garmendia, Carmen R. Beuzón{dagger}, Javier Ruiz-Albert{dagger} and David W. Holden

Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, UK

Correspondence
David W. Holden
d.holden{at}imperial.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The type III secretion system (TTSS) encoded by Salmonella typhimurium pathogenicity island 2 (SPI-2) is expressed after bacterial entry into host cells. The SPI-2 TTSS secretes the translocon components SseBCD, which translocate across the vacuolar membrane a number of effector proteins whose action is required for intracellular bacterial replication. Several of these effectors, including SifA and SifB, are encoded outside SPI-2. The two-component regulatory system SsrA–SsrB, encoded within SPI-2, controls the expression of components of the SPI-2 TTSS apparatus as well as its translocated effectors. The expression of SsrA–B is in turn regulated by the OmpR–EnvZ two-component system, by direct binding of OmpR to the ssrAB promoter. Several environmental signals have been shown to induce in vitro expression of genes regulated by the SsrA–B or OmpR–EnvZ systems. In this work, immunoblotting and flow cytometry were used to analyse the roles of SsrA–B and OmpR–EnvZ in coupling different environmental signals to changes in expression of a SPI-2 TTSS translocon component (SseB) and two effector genes (sifA and sifB). Using single and double mutant strains the relative contribution of each regulatory system to the response generated by low osmolarity, acidic pH or the absence of Ca2+ was determined. SsrA–B was found to be essential for the induction of SPI-2 gene expression in response to each of these individual signals. OmpR–EnvZ was found to play a minor role in sensing these signals and to require a functional SsrA–B system to mediate their effect on SPI-2 TTSS gene expression.


Abbreviations: FCS, fetal calf serum; GFP, green fluorescent protein; SCV, Salmonella-containing vacuole; SPI-2, Salmonella typhimurium pathogenicity island 2; TRSC, Texas red sulfonyl chloride; TTSS, type III secretion system

{dagger}Present address: Departamento de Biología Celular, Fisiología y Genética, Facultad de Ciencias, Universidad de Málaga, Campus Teatinos, Málaga 29071, Spain.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Type III secretion systems (TTSSs) mediate translocation of effector proteins from the bacterial cytoplasm of Gram-negative pathogens into the eukaryotic host cell. Once delivered into the host cell, effectors alter host cell functions to the advantage of the pathogen. TTSSs comprise a needle-like secreton for exporting proteins across the bacterial cell envelope, a translocon for transferring effector proteins into the host cell, several regulators that control gene transcription and protein secretion, chaperones that bind to and are needed for secretion of translocator and effector proteins, and the effector proteins themselves (Cornelis & Van Gijsegem, 2000; Hueck, 1998).

Serovars of Salmonella enterica encode two distinct virulence-associated TTSSs located within Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), which are involved in different aspects of S. enterica pathogenicity. The SPI-1 TTSS of S. enterica serovar Typhimurium (S. typhimurium) translocates at least eight effector proteins that control several processes, including host cell invasion, an apoptotic-like effect in macrophages, and trans-epithelial migration of neutrophils (Hersh et al., 1999; Zhou & Galán, 2001). The SPI-1 TTSS is expressed optimally in growth conditions that reflect those in the lumen of the small intestine, including low oxygen, high osmolarity and slight alkalinity (pH 8) (Bajaj et al., 1996). The S. typhimurium SPI-2 TTSS is required for systemic infection of mice, and intracellular replication in both macrophages and epithelial cells (Cirillo et al., 1998; Hensel et al., 1995, 1998; Ochman et al., 1996; Shea et al., 1996). SPI-2 TTSS gene expression is induced inside the host cell and requires the two-component regulatory system SsrA–B, also encoded within SPI-2 (Cirillo et al., 1998). SsrA–B controls the expression of genes encoding the components of the SPI-2 TTSS, as well as genes encoding SPI-2 effectors located both in SPI-2 and elsewhere in the chromosome (Beuzón et al., 2000; Brumell et al., 2003; Cirillo et al., 1998; Knodler et al., 2002; Miao & Miller, 2000; Worley et al., 2000). Two effector proteins, SlrP and SspH1, which have been shown to be translocated via both SPI-1 and SPI-2 TTSS, are expressed constitutively, in a SsrA–B-independent manner (Miao & Miller, 2000).

The expression of SsrA–B is regulated by the OmpR–EnvZ two-component system. OmpR binds directly to the ssrAB promoter (Lee et al., 2000). In Escherichia coli, OmpR–EnvZ has been shown to be responsible for both activation and repression of gene expression, in response to changes in osmolarity and pH (Heyde & Portalier, 1987). The OmpR–EnvZ system is required for Salmonella replication and survival within macrophages (Lee et al., 2000) and is necessary for full virulence in mice (Chatfield et al., 1991; Dorman et al., 1989).

Several studies have analysed SPI-2 TTSS gene expression in different conditions, thought to reflect the environment within the Salmonella-containing vacuole (SCV). Bacteria grown in different minimal media express SPI-2 TTSS genes when reaching stationary phase (Beuzón et al., 1999; Deiwick & Hensel, 1999; Deiwick et al., 1999; Lee et al., 2000; Miao et al., 2002). In addition, low osmolarity in the growth medium has been shown to play a role in the induction of SPI-2 TTSS gene expression (Lee et al., 2000). Low concentrations of Mg2+, Ca2+ or in growth media have also been reported to stimulate SPI-2 TTSS gene expression (Deiwick et al., 1999), although more recent studies have failed to reproduce the effect of low concentrations of Mg2+ (Lee et al., 2000; Miao et al., 2002). It has been shown that the SCV undergoes acidification to a pH between 4·0 and 5·0 (Rathman et al., 1996). Using transcriptional fusions of several SPI-2 genes to a gene encoding the green fluorescent protein (GFP), Cirillo et al. (1998) reported that inhibition of SCV acidification abolished SPI-2 TTSS gene expression inside the host cell. Using fusions to different reporters Lee et al. (2000) and Miao et al. (2002) found that acidic pH induced SPI-2 TTSS gene expression in vitro but Deiwick et al. (1999) found no significant differences in SPI-2 gene expression in response to pH changes.

In this study, we have determined the effect of [Ca2+], osmolarity and pH on SPI-2 gene expression and the relative influence exerted by the SsrA–B and OmpR–EnvZ two-component regulatory systems in this process. Our results show that the effects of these signals on SPI-2 TTSS gene expression are completely dependent on SsrA–B but only partially dependent on OmpR–EnvZ. Futhermore, the effect of OmpR–EnvZ on SPI-2 TTSS gene expression requires a functional SsrA–B system, which indicates that the effect of OmpR–EnvZ on SPI-2 gene expression is mediated through ssrAB.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
The bacterial strains used in this study are listed in Table 1. Strain HJG1 was constructed by P22-mediated transduction of an ompR1009 : : Tn10 mutation from strain HH182 to strain P3F4. Bacteria were grown in Luria–Bertani (LB) medium or in MES-buffered magnesium minimal medium (MgM) containing 170 mM MES at the corresponding pH (7·5 or 4·5), 5 mM KCl, 7·5 mM (NH4)2SO4, 0·5 mM K2SO4, 1 mM KH2PO4, 8 µM MgCl2, 38 mM glycerol and 0·1 % Casamino acids (Hmiel et al., 1986). Antibiotics were added at the following concentrations, as appropriate: ampicillin, 50 µg ml-1; kanamycin, 50 µg ml-1; and tetracycline, 25 µg ml-1. Bacteria were grown at 37 °C overnight with aeration.


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Table 1. Strains used in this study

 
Plasmids.
Plasmid pFVP25.1, carrying gfpmut3A under the control of a constitutive promoter, was introduced into bacterial strains for fluorescence visualization where indicated (Valdivia & Falkow, 1997). pID835 is a derivative of pFVP25, a vector carrying a promoterless gfpmut3A (Valdivia et al., 1996). pID835 contains a transcriptional fusion of the promoter region of the sifA region to the gfp gene. A fragment including 356 bp of the sifA promoter region and the sequence encoding the 5' -27 bp of sifA was amplified by PCR from 12023 genomic DNA using the primers SIFA-Prf1 (5'-CCGGAATTCTAATTGCGCAACGCTAAC-3') and SIFA-Prr1 (5'-CGCGGATCCAACCATTCCCTATAGTAA-3'). The 374 bp PCR product, containing terminal EcoRI and BamHI sites, was digested and ligated into pFVP25, generating pID835. pID836 is a derivative of pFVP25, containing a transcriptional fusion of the promoter region of sifB to the gfp gene. A 1·5 kb fragment containing the sifB promoter region and the sequence encoding the 5' -27 bp of the sifB gene was amplified by PCR from 12023 genomic DNA using the primers SifB-2 (5'-GGATCCTTTGAGCCTCCTCGCAGG-3') and SifB-5 (5'-CATATGACTCTGGTGATGAGCCTC-3'), and ligated into pCR2.1 topo vector (Novagen). The 1·5 kb fragment was excised from this intermediate plasmid by EcoRI digestion and then digested with DraI, generating an EcoRI–DraI 562 bp product, which was ligated into EcoRI–DraI-digested pFPV25, generating pID836.

Preparation of protein samples.
Bacterial cell densities were determined by measurement of the OD600. To ensure that protein from equal numbers of cells was analysed, in all experiments protein samples were adjusted to OD600 values such that each fraction from a 10 ml culture of OD600 0·6 was taken up in 100 µl protein-denaturing buffer for gel electrophoresis.

PAGE and Western analysis of proteins.
Protein samples were dissolved in the appropriate volume of protein-denaturing buffer containing 62·5 mM Tris/HCl pH 6·8, 2 % SDS, 5 % {beta}-mercaptoethanol, 10 % glycerol and 0·02 % bromophenol blue and held at 100 °C for 5 min. Proteins were immediately separated on a 12 % SDS-polyacrylamide gel (Laemmli, 1970). Proteins were transferred from gels to Immobilon-P membranes (Millipore) using a semi-dry blotting apparatus (Bio-Rad) with the buffer described by Kyhse-Andersen (1984). Westerns were developed using the ECL detection system under the conditions recommended by the manufacturer (Amersham Life Science). Rabbit anti-SseB (Beuzón et al., 1999) or anti-RecA (a gift from Kenji Adzuma, The Rockefeller University, New York, USA) polyclonal antibodies, or mouse monoclonal anti-GFP (Clontech) were used as primary antibodies. Donkey anti-rabbit or anti-mouse horseradish-peroxidase-conjugated antibodies (Amersham Life Science) were used as secondary antibodies. Affinity purification of anti-SseB antibody was performed using the method described by Ruiz-Albert et al. (2003).

Preparation of bacteria for flow cytometric analysis.
S. typhimurium 12023 wild-type and mutant strains carrying pID835 or pID836 plasmids were centrifuged at 4000 g, and pellets were resuspended in PBS. In each experiment, strain 12023 and strain 12023 carrying pFVP25.1 were used as negative and positive controls for fluorescence, respectively. For each sample, 105 cells were analysed on a FACS Calibur cytometer (Becton Dickinson). GFP was detected at 525 nm in the FL1 channel. Data were analysed with CellQuest software. Flow cytometric data were analysed as follows. The geometric mean of the fluorescence of each strain in three independent experiments was calculated. The fold increase in fluorescence of the ssrA, ompR, or ssrA ompR mutant strains versus that of the wild-type was calculated by dividing the geometric mean fluorescence of the wild-type strain by the geometric mean fluorescence of the mutants.

Antibodies and reagents.
Anti-Salmonella goat polyclonal antibody CSA-1 was purchased from Kirkegaard and Perry Laboratories and was used at a dilution of 1 : 400. Texas red sulfonyl chloride (TRSC)-conjugated donkey anti-goat antibody was purchased from Jackson Immunoresearch Laboratories and used at a dilution of 1 : 400.

Cell culture.
RAW 264.7 cells were obtained from ECACC (ECACC 91062702). HeLa cells (clone HtTA1) were kindly provided by Dr H. Bujard (Heidelberg, Germany). Cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10 % fetal calf serum (FCS) and 2 mM glutamine at 37 °C in 5 % CO2.

Bacterial infection of HeLa cells.
HeLa cells were seeded onto glass coverslips (12 mm diameter) in 24-well plates at a density of 5x104 cells per well, 24 h before infection. Bacteria were incubated for 16 h at 37 °C with aeration, diluted 1 : 33 in fresh LB broth and incubated in the same conditions for 3·5 h. Cultures were diluted in Earle's buffered salt solution (EBSS) pH 7·4 and added to the HeLa cells at a m.o.i. of 100 : 1. The infection was allowed to proceed for 15 min at 37 °C in 5 % CO2. The monolayers were washed once with DMEM containing FCS and 100 µg gentamicin ml-1 and incubated in this medium for 1 h, after which the gentamicin concentration was decreased to 16 µg ml-1.

Immunofluorescence.
For immunofluorescence, cell monolayers were fixed in 3·7 % paraformaldehyde in phosphate-buffered saline (PBS) pH 7·4, for 15 min at room temperature and washed three times in PBS. Antibodies were diluted in 10 % horse serum, 1 % bovine serum albumin, 0·1 % saponin in PBS. Coverslips were washed twice in PBS containing 0·1 % saponin, incubated for 30 min with primary antibodies, washed twice with 0·1 % saponin in PBS and incubated for 30 min with secondary antibodies. Coverslips were washed twice in 0·1 % saponin in PBS, once in PBS and once in H2O, and mounted on Mowiol. Samples were analysed using an Olympus BX50 fluorescence microscope or a Zeiss LSM510 confocal laser scanning microscope. For staining with anti-SseB antibody, cells were permeabilized for 10 min by incubation with 0·1 % Triton X-100 in PBS, prior to incubation with the antibody.

Bacterial infection of macrophages for flow cytometric analysis.
Macrophages were seeded at a density of 4x105 cells per well in 24-well tissue culture plates, 24 h before use. Bacteria were cultured at 37 °C with shaking until they reached an OD600 of 2·0. The cultures were diluted to an OD600 of 1·0 and opsonized in DMEM containing FCS and 10 % normal mouse serum for 20 min. Bacteria were added to the monolayers at a m.o.i. of 100 : 1, centrifuged at 170 g for 5 min at room temperature and incubated for 25 min at 37 °C in 5 % CO2. Macrophages were washed once with DMEM containing FCS and 100 µg ml-1 gentamicin and incubated in this medium for 1 h. The medium was replaced with DMEM containing FCS and 16 µg ml-1 gentamicin for the rest of the experiment. Bafilomycin A1 was added to cell monolayers 15 min prior to the addition of the bacteria, to a final concentration of 100 nM, where indicated. At 2 h after bacterial uptake, cells were lysed with 0·1 % Triton X-100 for 10 min, and used for flow cytometry.

Flow cytometric analysis of infected macrophages.
Infected macrophage lysates were resuspended in a mixture of 250 µl 0·1 % Triton X-100 and 250 µl PBS and kept on ice for immediate analysis. For each sample, 104 bacterial-sized particles were analysed on a FACS Calibur cytometer (Becton Dickinson). GFP was detected at 525 nm in the FL1 channel. Data were analysed with CellQuest software.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Effect of SsrA–B and OmpR–EnvZ on SPI-2 TTSS gene expression in infected cells
To analyse the expression of SPI-2 TTSS effector genes, transcriptional fusions of the sifA and sifB promoters to a gene encoding GFP were generated in the promoter trap vector pFPV25 (Valdivia & Falkow, 1997; Valdivia et al., 1996). SifA is translocated into the host cell by the SPI-2 TTSS (Brumell et al., 2001), and is necessary for the maintenance of the SCV membrane (Beuzón et al., 2000) and for Sif (Salmonella-induced filament) formation (Stein et al., 1996). SifB is also translocated by the SPI-2 TTSS and is expressed in vitro in an SsrA–B-dependent manner (Miao & Miller, 2000). Translocated SifB is localized on the SCV membrane and Sifs (Freeman et al., 2003); however, its function is unknown (Ruiz-Albert et al., 2002). The expression patterns of the PsifA : : gfp and PsifB : : gfp transcriptional fusions were analysed in HeLa cells infected with wild-type S. typhimurium harbouring the respective plasmids. Expression of PsifA : : gfp was only detected intracellularly, and was completely dependent on SsrA–B and partially dependent on OmpR–EnvZ, a pattern similar to that reported for a SifA : : GFP translational fusion (Beuzón et al., 2000). GFP could be detected as early as 2 h after invasion, and reached a peak 6 h after invasion (data not shown). Similarly, PsifB : : gfp expression was only detected intracellularly, and was completely dependent on SsrA–B (Fig. 1a). The intensity of fluorescence in bacteria carrying the PsifB : : gfp fusion was clearly higher than that in bacteria carrying PsifA : : gfp (data not shown). Approximately 50 % of ompR mutant bacteria carrying PsifB : : gfp displayed GFP expression inside the host cell, and the overall intensity of the fluorescence was noticeably lower than that displayed by wild-type bacteria carrying the same plasmid (data not shown). These results are similar to those reported for a SifA : : GFP translational fusion (Beuzón et al., 2000) and for a PssaH : : gfp transcriptional fusion (Cirillo et al., 1998).



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Fig. 1. (a) PsifB : : gfp expression in S. typhimurium wild-type (WT; 12023) and ssrA mutant strains. Confocal immunofluorescence analysis in HeLa cells 6 h after invasion with different strains carrying a plasmid-borne PsifB : : gfp transcriptional fusion. S. typhimurium was detected with goat anti-Salmonella and TRSC-conjugated donkey anti-goat antibodies (red). The right upper panel shows one extracellular bacterium not expressing GFP, and several GFP-positive intracellular bacteria. The lower panel shows lack of intracellular PsifB : : gfp expression in an ssrA mutant strain. Scale bars, 5 µm. (b) SseB was detected intracellularly in HeLa cells 6 h after invasion with wild-type but not with an sseB mutant strain. Bacteria were detected with goat anti-Salmonella and FITC-conjugated donkey anti-goat antibodies (green). SseB was detected with rabbit anti-SseB polyclonal affinity purified-antibody and TRSC-conjugated donkey anti-rabbit antibodies (red). Scale bars, 1 µm.

 
As an independent method to monitor the levels of another SPI-2-secreted protein, we used an anti-SseB antibody. SseB is a translocon component of the SPI-2 TTSS (Nikolaus et al., 2001), which is secreted in vitro in response to acidic pH (Beuzón et al., 1999). SseB is encoded within the SPI-2 sseABCDEFG operon, and displays the same expression profile as other components of the SPI-2 TTSS (Deiwick et al., 1999). Affinity-purified anti-SseB antibody was used to detect SseB by immunofluorescence microscopy in wild-type S. typhimurium after infection of host cells. SseB was detected in some wild-type bacteria 2 h after invasion, and was detected maximally at 6 h after invasion (Fig. 1b and data not shown). At this time point, SseB was detected in 50±2·1 % of wild-type, 0 % of ssrA mutant, and 19·5±3·9 % of ompR mutant bacteria (mean±SEM, n=100) in four independent experiments. These numbers are similar to those reported for SifA : : GFP (Beuzón et al., 2000) and ssaH : : gfp expression (Cirillo et al., 1998).

Regulation of sifA and sifB expression in vitro
Mg minimal (MgM) salts medium (see Methods) has been used to stimulate SPI-2 TTSS gene expression and to attempt to identify the signals that trigger expression of the SPI-2 TTSS inside the SCV (Deiwick & Hensel, 1999; Deiwick et al., 1999; Miao et al., 2002; Beuzón et al., 1999). Growth of bacteria in this medium results in a strong induction of SPI-2 TTSS expression. Therefore, this medium was used in this study as a basis to further investigate the signals stimulating SPI-2 gene expression. The pH of bacterial cultures after overnight growth in MgM medium buffered to pH 7·5 before inoculation remained unchanged.

Wild-type S. typhimurium carrying either PsifA : : gfp or PsifB : : gfp transcriptional fusions were grown overnight in MgM medium. Expression from each plasmid was detected by flow cytometry (Fig. 2a, b) and immunoblotting using an anti-GFP antibody (Fig. 3a, lower panel). As observed in infected cells (Fig. 1a), we found that expression driven by the sifB promoter was higher than that driven by PsifA (Fig. 2a, b, upper panels).



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Fig. 2. Flow cytometric analysis of Salmonella strains carrying PsifA : : gfp (a) or PsifB : : gfp (b) transcriptional fusions, grown overnight at 37 °C in MgM medium. Lines depict GFP expression in S. typhimurium wild-type (blue), ssrA (blue dashed), ompR (red) and ssrA ompR (red dashed) mutant strains. The black line shows a non-fluorescent negative control (S. typhimurium wild-type not carrying any transcriptional fusion). In each individual experiment 105 bacterial-sized particles were analysed. In the upper panels, the fluorescence intensity of each particle is reported on the x axis and the number of bacterial-sized particles is shown on the y axis. Lower panels show the fold increase in expression detected by flow cytometry, calculated as the geometric mean (n=3) of the fluorescence of the wild-type strain carrying the corresponding plasmids divided by the fluorescence of each mutant strain carrying the same plasmid.

 


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Fig. 3. (a) PsifA : : gfp and PsifB : : gfp expression by wild-type S. typhimurium carrying the corresponding plasmid grown in MgM medium or in MgM containing 200 µM Mg2+, overnight at 37 °C. GFP expression from PsifA : : gfp and PsifB : : gfp was detected by immunoblot analysis with mouse monoclonal anti-GFP and anti-mouse horseradish-peroxidase-conjugated antibodies (lower panel) and by flow cytometry (upper two panels). (b) Expression of SseB in response to changes in [Mg2+]. Immunoblot analysis of S. typhimurium wild-type 12023 grown in MgM medium or in MgM with 200 µM Mg2+, overnight at 37 °C. SseB was detected with rabbit polyclonal anti-SseB and anti-rabbit horseradish-peroxidase-conjugated antibodies (upper panel). The presence of equal amounts of protein in each sample was confirmed by immunoblot analysis using rabbit polyclonal anti-RecA and anti-rabbit horseradish-peroxidase-conjugated antibodies (lower panel).

 
PsifA : : gfp and PsifB : : gfp expression was then analysed in ssrA or ompR single mutant strains, and ssrA ompR double mutant strains after growth in the same culture conditions. The expression of PsifA : : gfp was reduced to levels undetectable by flow cytometry in any of these three mutant strains (Fig. 2a). PsifB expression levels were reduced but were reproducibly detectable in ssrA, ompR and ssrA ompR mutant strains (Fig. 2b). Expression of PsifB : : gfp was reduced more than tenfold in the ssrA mutant strain and twofold in the ompR mutant. The expression in the ssrA ompR double mutant strain was equivalent to that in the ssrA single mutant (Fig. 2b), indicating that the effect of OmpR is dependent on a functional SsrA–B system, in agreement with previous work (Lee et al., 2000), and that the signals that OmpR–EnvZ responds to are routed through SsrA–B. These results indicate that the SsrA–B and OmpR–EnvZ-dependent expression displayed in infected cells by the SPI-2 TTSS genes (Fig. 1) can be replicated in vitro by using MgM as bacterial growth medium.

Contribution of different environmental signals to SPI-2 TTSS gene expression in vitro
We next investigated the contribution of [Mg2+] to SPI-2 TTSS gene expression by growing bacterial strains in MgM medium containing either 8 µM or 200 µM MgCl2. Flow cytometry was carried out using wild-type bacteria harbouring either PsifA : : gfp or PsifB : : gfp fusions, after overnight growth in MgM medium at pH 7·5. No significant differences in fluorescence were detected for either fusion after growth in 8 µM or 200 µM MgCl2 (Fig. 3a, upper panel). Similarly, no significant differences were observed by immunoblotting either with an anti-GFP antibody using wild-type bacteria carrying either PsifA : : gfp or PsifB : : gfp fusions (Fig. 3a, lower panel), or with an anti-SseB antibody using wild-type bacteria (Fig. 3b).

Deiwick et al. (1999) reported that, after growth in MOPS-salts medium (O'Neal et al., 1994), the presence of 200 µM Mg2+ inhibited the expression of ssaB, and this effect could be reverted by lowering the [PO3-4]. However, in the presence of 8 µM Mg2+, ssaB expression was independent of [PO3-4]. In agreement with these results, no significant differences were observed in PsifA : : gfp or PsifB : : gfp expression in response to changes in [PO3-4] in medium containing 8 µM Mg2+ (data not shown). However, under our assay conditions, no inhibitory effect of high [Mg2+] was observed (Fig. 3a, b). This prevented us from confirming a possible effect of PO3-4 starvation in reverting an inhibitory effect of high [Mg2+].

Removal of 38 mM glycerol from the medium had no significant effect on sifA : : gfp or sifB : : gfp expression, when analysed by flow cytometry (data not shown). Similarly, reducing [(NH4)2SO4] in the growth medium by 10- or 100-fold did not have a significant effect on sifA : : gfp or sifB : : gfp expression (data not shown).

Together, these results indicate that changes in the concentrations of Mg2+, PO3-4, glycerol and (NH4)2SO4 have no significant effect on SPI-2 gene expression, after overnight growth in MgM medium.

The absence of Ca2+ leads to SPI-2 TTSS gene expression via SsrA–B
It has been shown previously that the absence of Ca2+ in Tris-buffered MgM medium induces expression from the SPI-2 promoters PssaB and PsseA (Deiwick et al., 1999). Consistent with these results, the fluorescence of wild-type bacteria carrying either PsifA : : gfp or PsifB : : gfp increased by six- to eightfold and eightfold, respectively, after growth in MgM medium, compared to the expression after growth in MgM medium containing 2 mM Ca2+ (data not shown and Fig. 4b). Similarly, SseB was undetectable in wild-type bacteria grown overnight in MgM medium in the presence of 2 mM Ca2+, but was detected after growth in MgM medium without Ca2+ (Fig. 4c).



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Fig. 4. (a) PsifA : : gfp expression in S. typhimurium wild-type, ssrA, ompR, or ssrA ompR mutant strains grown in MgM medium or in MgM with 2 mM Ca2+, overnight at 37 °C. GFP expression from PsifA : : gfp was detected by immunoblot analysis using mouse monoclonal anti-GFP and anti-mouse horseradish-peroxidase-conjugated antibodies. The presence of equal amounts of protein in each sample was confirmed as described in Fig. 3(a). (b) PsifB : : gfp expression in S. typhimurium wild-type, ssrA, ompR, and ssrA ompR mutant strains grown in MgM medium or in MgM with 2 mM Ca2+, overnight at 37 °C. GFP expression for PsifB : : gfp was detected by flow cytometry. The lower panel shows the fold increase in expression detected, calculated as the geometric mean (n=3) of the fluorescence of the wild-type and mutant strains carrying PsifB : : gfp, grown in MgM, divided by that of the corresponding strain grown in MgM with 2 mM Ca2+. (c) Expression of SseB in response to changes in [Ca2+]. Immunoblot analysis of S. typhimurium 12023 grown in MgM medium or in MgM with 2 mM Ca2+, overnight at 37 °C. SseB was detected with rabbit polyclonal anti-SseB and anti-rabbit horseradish-peroxidase-conjugated antibodies.

 
To determine which regulatory system mediates the effect of low [Ca2+], we analysed PsifA : : gfp expression in ssrA or ompR single mutant strains, and ssrA ompR double mutant strains, after growth in MgM medium with or without 2 mM Ca2+. Since expression of PsifA : : gfp in the mutant strains was below the level of detection by flow cytometry, the protein levels were determined by immunoblot analysis. The higher level of sifA : : gfp expression in low [Ca2+] was completely abolished in the ssrA single mutant and the ssrA ompR double mutant strains (Fig. 4a). This indicates that induction of sifA expression in response to the absence of Ca2+ is dependent on the presence of SsrA–B. In the ompR mutant sifA : : gfp expression levels were higher in low [Ca2+] (Fig. 4a), although it was not possible to determine if the level of induction was equivalent to that of the wild-type strain. Similarly, when analysed by flow cytometry, the effect of 2 mM Ca2+ on sifB : : gfp expression was completely abolished in both the ssrA single mutant and the ssrA ompR double mutant strains, whereas in the ompR mutant sifB : : gfp expression was decreased 3·5-fold (Fig. 4b). Together, these results indicate that the response to low [Ca2+] is mediated mainly through SsrA–B. Although not as important as SsrA–B, OmpR–EnvZ is necessary for full induction of SPI 2 TTSS gene expression in response to low [Ca2+].

Low osmolarity induces SPI-2 TTSS gene expression via SsrA–B
It has been shown previously that high osmolarity (generated by the presence of 20 % sucrose or 0·5 M NaCl) in MgM medium represses expression from the SPI-2 promoter PssaH (Lee et al., 2000). S. typhimurium is not able to use sucrose as a carbon source, but the presence of Casamino acids allows normal growth of bacterial cells in MgM medium containing 20 % sucrose. Expression of PsifA : : gfp and PsifB : : gfp was eight- and tenfold higher, respectively, in MgM medium than in the same medium containing 20 % sucrose (Fig. 5a, upper panel, and 5b). The addition of the osmoprotectant glycine betaine partially prevented the inhibitory effect of 20 % sucrose on both PsifA : : gfp and PsifB : : gfp expression, showing that the effect of sucrose is mainly osmotic (Fig. 5a, upper panel, and 5b). SseB levels were also higher in the absence than in the presence of 20 % sucrose (Fig. 5c).



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Fig. 5. (a) PsifA : : gfp expression by S. typhimurium wild-type, ssrA, ompR, and ssrA ompR mutant strains grown in MgM medium or in MgM with 20 % sucrose, overnight at 37 °C. GFP expression from PsifA : : gfp was detected by flow cytometry (wild-type strain in upper panel) and by immunoblot analysis using mouse monoclonal anti-GFP and anti-mouse horseradish-peroxidase-conjugated antibodies (lower panel). The upper panel shows the fold increase in expression detected by flow cytometry, calculated as the geometric mean (n=3) of the fluorescence of the wild-type strain carrying PsifA : : gfp fusion grown in MgM (left column) or in MgM with 20 % sucrose and glycine betaine (right column) divided by the fluorescence of the same strain grown in MgM with 20 % sucrose. (b) PsifB : : gfp expression in S. typhimurium wild-type, ssrA, ompR, and ssrA ompR mutant strains grown in MgM medium or in MgM with 20 % sucrose, overnight at 37 °C. PsifB : : gfp expression was detected by flow cytometry. The fold increase in expression was calculated as the geometric mean (n=3) of the fluorescence of the wild-type and mutant strains carrying PsifB : : gfp grown in MgM divided by that of the corresponding strain grown in MgM with 20 % sucrose. The right-hand column shows the fluorescence of the wild-type strain carrying PsifB : : gfp grown in MgM with 20 % sucrose and glycine betaine divided by the fluorescence of the wild-type strain grown in MgM with 20 % sucrose. (c) Expression of SseB in response to changes in osmolarity. Immunoblot analysis of S. typhimurium wild-type 12023 grown in MgM medium or in MgM with 20 % sucrose, overnight at 37 °C. SseB was detected with rabbit polyclonal anti-SseB and anti-rabbit horseradish-peroxidase-conjugated antibodies (upper panel).

 
To determine which regulatory system mediates the osmotic effects of sucrose, the ssrA or ompR single mutant, and ssrA ompR double mutant strains carrying PsifA : : gfp were grown in MgM medium with or without 20 % sucrose and then analysed by immunoblotting with the anti-GFP antibody. The higher level of sifA : : gfp expression in low osmolarity was completely abolished in the ssrA single mutant and the ssrA ompR double mutant strains (Fig. 5a, lower panel). However, the decrease in PsifA : : gfp expression in the presence of 20 % sucrose could still be observed in an ompR mutant (Fig. 5a, lower panel). Flow cytometry of single and double mutant strains carrying PsifB : : gfp showed that the higher level of PsifB : : gfp expression at low osmolarity was abolished in the ssrA single mutant and ssrA ompR double mutant strains. However, in the ompR mutant strain, the osmotic effect of sucrose accounted for a sixfold difference in gene expression (Fig. 5b). These results indicate that the effect of osmolarity on the expression from sifA and sifB promoters is mediated predominantly through SsrA–B, and that OmpR–EnvZ plays a minor role in sensing this signal.

It has been shown that S. typhimurium reacts to a sudden increase in osmolarity by taking up large amounts of K+ from the environment (Kempf & Bremer, 1998). Jung et al. (2001) showed that the EnvZ-autokinase activity of purified and reconstituted EnvZ is stimulated in the presence of increasing [KCl] and the amount of phosphorylated OmpR in the reconstituted signal cascade increases over time in the presence of KCl, suggesting that [K+] in the medium could act as the signal detected by OmpR–EnvZ when changes in osmolarity occur. Although the [K+] in the SCV is not known, [K+] in Staphylococcus aureus-containing vacuoles in neutrophils has been reported to be in the 200–300 mM range (Reeves et al., 2002). Therefore, we investigated if changes in [K+] affect the expression of sifA, sifB and sseB. However, no effects comparable to those caused by changes in osmolarity were detected. The increase of [KCl] from 5 mM to 200 mM caused only a minor increase in the level of SseB detected by immunoblotting (data not shown). No significant differences were observed in PsifA : : gfp or PsifB : : gfp expression either by flow cytometry or by immunoblotting using an anti-GFP antibody, at 5 mM or 200 mM KCl (data not shown). Despite these results, we cannot rule out a role for [K+] in mediating osmolarity-dependent activation of OmpR–EnvZ, since the extracellular increase of [K+] might not result in an increase in its uptake into the periplasm.

SsrA–B and OmpR–EnvZ are both required to mediate the effect of acidic pH on SPI-2 TTSS gene expression
Flow cytometry and immunoblot experiments were performed to analyse the effect of pH on PsifA : : gfp and PsifB : : gfp expression, by comparing the expression of each gene in bacterial cultures grown in MgM medium at pH 7·5 or at pH 4·5. However, the results obtained were too variable to allow any conclusions to be drawn. On the other hand, levels of SseB were consistently higher in MgM medium at pH 4·5 than at pH 7·5 (Fig. 6c). The effect of pH on the expression of sifA : : gfp and sifB : : gfp was then analysed in infected macrophages, where acidification of the SCV can be prevented by treatment with bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, which acidifies endosomal and lysosomal compartments (Cirillo et al., 1998). PsifA : : gfp and PsifB : : gfp expression by intracellular wild-type bacteria was strongly inhibited in macrophages pre-treated with bafilomycin A1, as shown by immunofluorescence and flow cytometry, respectively (Fig. 6a, b). Flow cytometric analysis showed that the expression of PsifB : : gfp was twofold higher in intracellular bacteria inside non-treated macrophages than inside those treated with bafilomycin A1 (Fig. 6b). These results are in agreement with those obtained by Cirillo et al. (1998) for vacuole acidification-dependent induction of ssaJ, sscB and spiA (ssaC) expression. The absence of detectable PsifA : : gfp expression by both microscopy and flow cytometry when the fusion construct was carried by intracellular ssrA, ompR, and ssrA ompR mutant bacteria prevented further analysis of this promoter.



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Fig. 6. (a) PsifA : : gfp expression in bafilomycin A1-treated or untreated macrophages. Bafilomycin A1-treated or untreated macrophages were infected with S. typhimurium wild-type strain 12023 carrying the PsifA : : gfp transcriptional fusion and gfp expression was analysed by confocal immunofluorescence microscopy of macrophages 2 h after uptake. S. typhimurium was detected with goat anti-Salmonella and TRSC-conjugated donkey anti-goat antibodies (red). Scale bar, 5 µm. (b) PsifB : : gfp expression in bafilomycin A1-treated (blue lines) or untreated (red lines) macrophages. Bafilomycin A1-treated or untreated macrophages were infected with S. typhimurium wild-type, ssrA, ompR, ssrA ompR mutant strains carrying the PsifB : : gfp transcriptional fusion. At 2 h after uptake, intracellular bacteria were released by treating the samples with 0·1 % Triton X-100 for 10 min and their fluorescence was measured by flow cytometry. The fold increase in expression was calculated as the geometric mean (n=3) of the fluorescence of the wild-type and mutant strains carrying PsifB : : gfp from untreated cells divided by that of the corresponding strain released from bafilomycin A1-treated macrophages. (c) Expression of SseB in response to changes in pH. Immunoblot analysis of S. typhimurium 12023 grown in MgM medium (pH 7·5) or in MgM pH 4·5 overnight at 37 °C. SseB was detected with rabbit polyclonal anti-SseB and anti-rabbit horseradish-peroxidase-conjugated antibodies.

 
Flow cytometric analysis showed that PsifB : : gfp expression in ssrA and ompR mutant bacteria within non-treated macrophages was, respectively, 1·2-fold and 1·5-fold higher than that displayed by the same bacterial strains within bafilomycin A1-treated macrophages (Fig. 6b). The effect of bafilomycin A1 was completely abolished only in the ssrA ompR double mutant strain. These results suggest that the induction of sifB expression by acidic pH inside the vacuole is mediated by both SsrA–B and OmpR–EnvZ.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we have attempted to clarify the nature of the environmental signals that induce SPI-2 TTSS gene expression, and to determine whether these signals are transmitted through the SsrA–B or the OmpR–EnvZ regulatory system. We found that low osmolarity and absence of Ca2+ are the major signals affecting SPI-2 TTSS gene expression when bacteria are grown in MgM medium. Our results support those previously reported by Miao et al. (2002) and Lee et al. (2000) but differ from those of Deiwick et al. (1999), who reported that in MgM medium buffered with Tris/HCl or Bistris/HCl, [Mg2+] has an effect on sseA and ssaB expression. Also, we cannot exclude the possibility that other environmental signals affect SPI-2 gene expression. For example, [Fe2+] appears to have a role in down-regulating the expression of ssrA, sseA and sseJ by an unknown iron-responsive regulatory system (Zaharik et al., 2002).

The main finding of this work is that the effects on SPI-2 gene expression caused by the absence of Ca2+ and low osmolarity are transmitted predominantly through SsrA–B, whereas acidic pH is sensed by both SsrA–B and OmpR–EnvZ (Fig. 7). Lee et al. (2000) reported that the effect of osmolarity and acidic pH on ssaH transcription was decreased in an ompR mutant. In agreement with these results, we found that OmpR–EnvZ is required for the full expression of SPI-2 genes in response to these signals. Furthermore, this effect fully depends on a functional SsrA–B system, since the phenotype of an ssrA ompR double mutant in all three conditions is identical to that of the ssrA single mutant. These results indicate that the effect of OmpR–EnvZ on the expression of SPI-2 genes, other than ssrAB, is not a result of direct binding of OmpR to their promoters but is indirect, probably as a consequence of its binding to the ssrAB promoter (Lee et al., 2000).



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Fig. 7. Model for Salmonella SPI-2 TTSS gene expression regulation inside host cells. The OmpR–EnvZ two-component regulatory system has a minor role in mediating the response to the absence of Ca2+ and low osmolarity (dashed lines), but a greater role in mediating the response to acidic pH (solid line). The activated form of OmpR (OmpR*) binds to the ssrAB promoter and activates the transcription of ssrA and ssrB genes. SsrA, the sensor element of the SsrA–B two-component system, detects the absence of Ca2+, acidic pH and low osmolarity inside the vacuole directly or via unknown sensor(s), and activates SsrB. SsrB activates the expression of SPI-2-related genes, both inside and outside the pathogenicity island.

 
Our results indicate that SsrA–B, a single two-component regulatory system, can mediate the effect of three physiologically disparate environmental signals: acidic pH, low osmolarity and absence of Ca2+. This is reminiscent of the BvgAS two-component regulatory system, which controls the Bordetella pertussis virulence regulon in response to diverse environmental signals. Transcription of bvgAS-activated genes is almost completely abolished by a variety of environmental factors, which include the levels of sulfate anion and nicotinic acid, and the temperature of the growth medium (Miller et al., 1992; Uhl & Miller, 1994). Sequence similarity searches of the protein domain databases reveal that SsrA, like BvgS (Beier et al., 1995), is an unorthodox two-component sensor protein, since it contains a C-terminal region similar to the response regulator receiver domain (represented by the Interpro database entry IPR001789), usually found only in the response regulators.

Whether SsrA can sense different environmental signals directly is an open question. It is formally possible that SsrB could be activated independently from SsrA. It is also possible that at least one of the signals is sensed by another sensor–regulator and that this is then linked through SsrA–B. This is the case for the PmrA–B-regulated genes, whose expression is modulated by the [Mg2+] and [Fe3+] in the environment by a regulatory cascade of two-component regulatory systems, where the first one (PhoP–Q) senses [Mg2+] and the second (PmrA–B) senses [Fe3+] (Groisman, 2001). Supporting this notion, the expression of sifB : : gfp in MgM medium in an ssrA single or ssrA ompR double mutant strain remained eightfold higher than in LB, where sifB : : gfp expression is severely repressed (data not shown), suggesting that additional regulatory system(s) contribute to sifB expression. It was initially proposed that PhoP–Q (Groisman, 2001), required for intra-macrophage replication and systemic growth within the mouse (Fields et al., 1986), could be involved in the control of SPI-2 gene expression (Deiwick et al., 1999; Worley et al., 2000). However, it has been recently shown that PhoP–Q and the SPI-2 TTSS are functionally independent (Beuzón et al., 2001; Miao et al., 2002). One possible candidate is the transcriptional regulator SlyA, which is required for virulence and survival in macrophages (Libby et al., 1994). slyA mutants are sensitive to oxidative products of the respiratory burst (Buchmeier & Libby, 1997). Another candidate for the additional regulation of SPI-2 TTSS genes is the alternative sigma factor RpoE ({sigma}E). rpoE mutant strains are highly attenuated in mice (Humphreys et al., 1999). Although able to invade both macrophage and epithelial cell lines normally, the rpoE mutant is defective in its ability to survive and proliferate in both cell lines (Humphreys et al., 1999), and has also an increased sensitivity to the respiratory burst (Testerman et al., 2002).

What is the physiological significance of the roles of Ca2+, low osmolarity and acidic pH in regulating SPI-2 TTSS gene expression? Using fluorescence lifetime imaging microscopy, it has been shown that inside macrophage lysosomes [Ca2+] is at least five times lower (400 µM) than the concentration outside the cell (2 mM) (Christensen et al., 2002). Although the [Ca2+] in the lumen of the SCV is unknown, the strong repression of SPI-2 TTSS gene expression observed in vitro at a [Ca2+] equivalent to that found outside the cell (2 mM) could explain the absence of SPI-2 gene expression in extracellular bacteria (Beuzón et al., 2000). Low osmolarity represses SPI-1 TTSS gene expression (Bajaj et al., 1996), whereas it induces SPI-2 TTSS gene expression in vitro (this work; Lee et al., 2000). An opposite regulation by osmolarity on SPI-1 TTSS gene expression (expressed extracellularly and required for invasion) and SPI-2 TTSS (expressed intracellularly and required for intracellular replication) could help to ensure that these functionally distinct systems are expressed independently and only when and where they are required during the infection process. It has also been shown that the majority of vacuoles containing S. typhimurium acidify from pH 6·0 to between pH 4·0 and 5·0 within 60 min after formation (Rathman et al., 1996). Although it is clear that acidic pH induces SPI-2 TTSS-mediated secretion in vitro, the effect of acidic pH on inducing SPI-2 TTSS gene expression has been controversial (Beuzón et al., 1999; Deiwick et al., 1999; Lee et al., 2000; Miao et al., 2002). In this study, we found it impossible to ascertain whether acidic pH has a significant effect on SPI-2 TTSS gene expression in vitro. However, in infected macrophages, acidic pH has a clear effect on PsifB : : gfp expression (Fig. 6), confirming the requirement of vacuolar acidification for SPI-2 gene expression reported by Cirillo et al. (1998). If the effect of pH on SPI-2 TTSS expression is not as strong as its effect on secretion, or the effective range of action is very narrow, it is possible that, even in pH-controlled in vitro conditions, undetectable variations of pH from experiment to experiment could result in a lack of reproducibility which might explain the different results obtained (Deiwick et al., 1999; Lee et al., 2000; Miao et al., 2002). In conclusion, [Ca2+], osmolarity and pH could account for SPI-2 TTSS intracellular expression in vivo. These signals are predominantly transmitted through SsrA–B and may involve additional sensing proteins and regulators of the SPI-2 system.


   ACKNOWLEDGEMENTS
 
We thank Kate Unsworth for the critical reading of the manuscript and Suzana Salcedo and Aaron Rae for their help with the flow cytometry. J. Garmendia is recipient of a long-term postdoctoral EMBO fellowship. This research was also supported by a grant from the Medical Research Council (UK).


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Received 4 April 2003; revised 20 June 2003; accepted 20 June 2003.