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J. Biol. Chem., Vol. 279, Issue 48, 49804-49815, November 26, 2004

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Expression and Secretion of Salmonella Pathogenicity Island-2 Virulence Genes in Response to Acidification Exhibit Differential Requirements of a Functional Type III Secretion Apparatus and SsaL^*

Brian K. Coombes, Nat F. Brown, Yanet Valdez, John H. Brumell¶, and B. Brett Finlay||**

From the Michael Smith Laboratories and the Departments of ^||Biochemistry and Molecular Biology, ^**Microbiology, and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Received for publication, April 19, 2004 , and in revised form, August 25, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Salmonella pathogenicity island (SPI)-2 is pivotal to the intracellular survival of Salmonella and for virulence in mammals. SPI-2 encodes virulence factors (called effectors) that are translocated into the host cell, a type III secretion apparatus and a two-component regulatory system that regulates intracellular expression of SPI-2. Salmonella SPI-2 secretion activity appears to be induced in response to acidification of the vacuole in which it replicates. Here we show that the expression of the SPI-2 proteins, SseB and SseD (filament and pore forming components of the secretion apparatus, respectively) in response to acidification requires an intact secretion system and SsaL, a Salmonella homologue of SepL, a regulator required for type III-dependent secretion of translocators but not effectors in attaching and effacing gastrointestinal pathogens. We show that the expression of SPI-2-encoded effectors is acid-regulated but can be uncoupled from the expression of filament and translocon components, thus showing a differential requirement of SsaL for expression. The secretion and translocation of SPI-2-encoded effectors requires SsaL, but SsaL is dispensable for the secretion of SPI-2 effectors encoded in other pathogenicity loci, suggesting a secretion regulation function for SsaL. Further, we demonstrate that the differential expression of adjacent genes within the sseA operon (sseD and sseE) occurs at the transcriptional level. These data indicate that a Salmonella SPI-2 activation state is achieved by an acidregulated response that requires SsaL. These data also suggest the existence of a previously unrecognized regulatory element within SPI-2 for the "effector operon" region downstream of sseD that might demarcate the expression of translocators and effectors.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Intracellular survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) requires a set of virulence proteins encoded in the chromosomal pathogenicity island designated Salmonella pathogenicity island (SPI)¹-2 (1, 2). SPI-2 is a virulence locus that encodes a type III secretion system (TTSS) and related proteins, called effectors, which are substrates of this secretion apparatus. Infection of macrophages by Salmonella activates the SPI-2 virulence locus, which allows Salmonella to establish a replicative vacuole, called the Salmonella-containing vacuole (SCV), inside host cells. SPI-2-dependent activities are responsible for SCV maturation along the endosomal pathway to prevent bacterial degradation in phagolysosomes, for interfering with trafficking of NADPH oxidase-containing vesicles to the SCV (3), remodeling of host cell microfilaments (4, 5) and microtubule networks (6, 7), both of which play a role in maintaining the integrity of the SCV membrane and directing SCV traffic. As such, the regulation of SPI-2 is integral to Salmonella pathogenesis.

SPI-2 TTSS substrates share a similar temporal pattern of expression within host cells and are activated in response to environmental cues presumably sensed by the bacteria in the SCV lumen. Within SPI-2, these substrates include effectors, SseB (a component of the oligomeric filament structure of the type III apparatus), SseC, and SseD (the pore-forming translocation complex, or translocon) (8). To date, only three SPI-2-encoded effectors, SseF, SseG (9, 10), and probably SsaB (SpiC) (11), are documented to be translocated into host cells in an SPI-2-dependent fashion, although SPI-2 substrates are also encoded outside SPI-2 in other pathogenicity islands (12) and in lysogenic prophages (13), which are not genetically linked to SPI-2. The coordinated expression of SPI-2 secretion substrates is controlled by the SPI-2-encoded two-component regulatory system SsrA/SsrB (1, 14) and the upstream regulators OmpR/EnvZ, which modulate the expression of the ssrAB operon (15). Once activated, SsrB acts on multiple promoters in SPI-2 and in various regions of the genome (16, 17) to induce synthesis of SPI-2 translocator and effector substrates. Several open reading frames encoded within SPI-2 remain uncharacterized, including ssaL, encoding a protein with homology to the locus of enterocyte effacement (LEE)-encoded sepL of the attaching and effacing pathogens enterohemorrhagic Escherichia coli (EHEC) (18), enteropathogenic E. coli (EPEC), and Citrobacter rodentium (19). We recently identified sepL in a genetic screen and showed it is required for type III secretion in C. rodentium of the translocon complex, EspA, -B, and -D, but not for the secretion of the LEE-encoded effector Tir (20). Interestingly, ssaL and sepL contain no strong homologues in other pathogenic bacteria with type III secretion systems indicating that they probably are not part of the TTSS core complex, which is generally well conserved among type III-containing pathogens (21).

Previous work on the environmental cues within the host cell that activate SPI-2 gene expression and type III secretion has implicated Mg²⁺ and ion limitation and pH (15, 17, 22, 23). Because the pH of the SCV falls below 5.5 within 20 min after its formation inside infected host cells (24), it is possible that this environmental cue plays a prominent regulatory role in SPI-2 induction. In vitro conditions resembling the intracellular environment of the SCV such as low pH of minimal medium activate the secretion activity of SPI-2 (22, 25), but less is known about how acidic pH regulates SPI-2 gene expression. In two previous studies, the pH of minimal medium did not have an effect on the expression of SPI-2 genes (22) or on a SPI-2 substrate encoded within a prophage outside of SPI-2 (17). However, it was also reported that acidic pH of minimal medium activated the ssrA promoter in wild-type Salmonella (15), highlighting an apparent discrepancy given that SsrA and SsrB are required for SPI-2 gene expression.

To examine the way in which Salmonella responds to acidification of the vacuole in which it resides, we analyzed the expression and secretion of SPI-2 effectors and translocators under pH conditions that mimic those of the SCV. We demonstrate that expression and secretion of both translocators and effectors occurs only in acidic minimal medium and not in minimal medium at neutral pH. A Salmonella strain with a mutation in ssaR (a conserved type III apparatus component) is deficient in not only the secretion of effectors and translocators but also in the expression of translocators at acidic pH. A Salmonella strain deficient in ssaL, an uncharacterized SPI-2 open reading frame with homology to the secretion regulator sepL of attaching and effacing pathogens, displays a phenotype distinct from that of the ssaR mutant in that it restricts the expression and secretion of translocators encoded within SPI-2 yet is dispensable for secretion of SPI-2 effectors encoded in other pathogenicity loci. We also demonstrate that, after acidification, a functional type III secretion system and SsaL are required for optimal activation of the sseA promoter, which controls the expression of the SPI-2 filament and pore-forming components, but show that expression of the downstream effectors is differentially regulated with respect to type III secretion and SsaL.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bacterial Strains and Mutant Construction—S. Typhimurium strain SL1344 was used as the wild-type strain throughout this study, and all mutants used in these experiments are isogenic derivatives of SL1344. Strains used in this work are listed in Table I. S. Typhimurium and E. coli strains were routinely cultured in LB broth supplemented with the appropriate antibiotics to maintain plasmids as required. A nonpolar, unmarked, in-frame deletion of ssaL was generated by allelic exchange from a counter-selectable suicide vector expressing SacB (26). First, the ssaL open reading frame and 1 kb of flanking genomic DNA upstream and downstream of ssaL were amplified by PCR using the oligonucleotide primers BKC45 (5'-CCA GAG TAT CGG CAA TTG CTT-3') and BKC46 (5'-AAC AGC CTC ACT CAT CGA CAT-3') to generate a 3038-bp DNA fragment. This fragment was cloned into pCR2.1 (Invitrogen) to generate a plasmid template that was amplified by inverse PCR using BKC47 (5'-ACG CGT CGA CCA TCG CTA CCT CTT TTA TCT TCA C-3') and BKC48 (5'-ACG CGT CGA CAA GTC GGT TTT ATT CTG ATA CCT GGC-3', SalI sites underlined). This PCR product was digested with SalI and then ligated to generate an internal, in-frame deletion allele of ssaL that eliminated the coding region for amino acids 9–333, which was confirmed by DNA sequencing. The ssaL deletion allele was cloned into the unique XbaI and SacI sites of pRE112 (27) and transformed into E. coli SM10 pir (28) to generate a donor strain for conjugation. pRE112-ssaL was conjugated into S. Typhimurium SL1344 and merodiploid colonies were isolated, grown for 6 h in LB broth without antibiotic selection, diluted, plated onto agar containing 1% (w/v) Tryptone, 0.5% (w/v) yeast extract, 5% (w/v) sucrose, and incubated at 30 °C overnight. Sucrose-resistant colonies were selected, and the presence of the ssaL deletion was confirmed by PCR, restriction enzyme analysis, and DNA sequencing.

Recombinant SPI-2 Proteins and Generation of Antibodies—Polyclonal antibodies to SseB, SseD, SseE, and SseG were generated by repeated immunization of New Zealand White rabbits with 1 mg of recombinant glutathione S-transferase fusions of each protein. Each glutathione S-transferase fusion was constructed by PCR amplification of the effector gene from genomic DNA of S. Typhimurium SL1344 using the following primers: SseB, forward 5'-GCA GGA TCC ATG TCT TCA GGA AAC ATC TTA TGG-3', reverse 5'-CGT GTC GAC TCA TGA GTA CGT TTT CTG CGC TAT-3'; SseD, forward 5'-GCA GGA TCC ATG GAA GCG AGT AAC GTA GCA CTG-3', reverse 5'-CGT GTC GAC TTA CCT CGT TAA TGC CCG GAG TAT-3'; SseE, forward 5'-GCA GGA TCC ATG GTG CAA GAA ATA GAG CAA TGG-3', reverse 5'-CGT GTC GAC TTA AAA ACG TCG CTG GAT AAG ATG-3'; SseG, forward 5'-GCA GGA TCC ATG AAA CCT GTT AGC CCA AAT GCT-3', reverse 5'-CGT GTC GAC TTA CTC CGG CGC ACG TTG TTC TGG-3'. After digestion with BamHI and SalI (sites underlined above), the PCR product was cloned into pGEX6P-1 (Amersham Biosciences). This plasmid was transformed into the BL21 strain of E. coli, and overexpression of recombinant fusion proteins was accomplished with 1 mM isopropyl 1-thio--D-galactopyranoside for 3 h (1 h for expression of SseG). SseB was purified using glutathione-coupled beads (Sigma) according to standard protocols (29). Purification of SseD, SseE, and SseG was performed using the Sarkosyl lysis procedure described by Frangioni and Neel (30). Raw antisera were affinity-purified using Sepharose beads (Amersham Biosciences) with covalently coupled antigen. Non-specific cross-reactivity of the antibodies was minimized by incubation with acetone powders of the BL21 strain that was used to overexpress the recombinant fusion proteins.

Cell Culture—HeLa cells were maintained in Dulbecco's modified Eagle's medium (HyClone, Logan, UT) supplemented with 10% fetal calf serum. For immunofluorescence studies, HeLa cells were seeded onto 1-cm sterile glass coverslips in 24-well tissue culture dishes and incubated for 18 h prior to infection. For gentamicin protection assays, RAW 264.7 murine macrophages were used between passages 10 and 15 and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. RAW 264.7 cells were seeded in 24-well culture dishes 18 h prior to infection. All cell lines were cultured at 37 °C in 5% CO₂.

Analysis of Salmonella Mutants in Mice—Female BALB/c mice (6–8 weeks old) were purchased from Harlan Laboratories (Indianapolis, IN). Mice were housed in sterilized, filter-top cages under specific pathogen-free conditions at the University of British Columbia Animal Facility. The protocols used here were in direct accordance with animal care guidelines as outlined by the University of British Columbia's Animal Care Committee and the Canadian Council on the Use of Laboratory Animals. Salmonella cultures were grown overnight in LB broth and then diluted in phosphate-buffered saline to give 1.6 x 10⁵ cfu/ml. Groups of mice (n = 5) were infected by intraperitoneal injection with 0.3 ml of diluted bacterial suspension, followed by daily monitoring throughout the study period. Mice that showed signs of extreme distress were euthanized.

Gentamicin Protection Assays—RAW 264.7 cells were infected with opsonized stationary phase bacteria as described previously (31). At 2 and 21 h after infection, gentamicin-treated cells were washed with phosphate-buffered saline and then lysed in 0.25 ml of 1% Triton X-100, 0.1% SDS in phosphate-buffered saline. Lysates were diluted in phosphate-buffered saline and plated onto LB agar followed by incubation at 37 °C. Colonies were enumerated and expressed as colony forming units (cfu)/ml. The -fold increase in the number of intracellular bacteria was determined by dividing the cfu values at 21 h by the cfu values at 2 h post infection for each condition.

In Vitro Secretion Assays—In vitro culture conditions were developed based on minimal medium used to induce expression and secretion of SPI-2 genes (8, 22). Salmonella strains were grown overnight in LB broth, washed twice in low phosphate, low magnesium-containing medium (LPM) and then inoculated at a 1:50 dilution in 3 ml of LPM medium at either pH 7.0 or 5.8. The composition of LPM medium was 5 mM KCl, 7.5 mM (NH₄)₂SO₄, 0.5 mM K₂SO₄, 38 mM glycerol (0.3% v/v), 0.1% casamino acids, 8 µM MgCl₂, 337 µM , 100 mM Tris-HCl (for titration to pH 7.0), or 80 mM MES (for titration to pH 5.8). Cultures were grown at 37 °C with shaking for 4–6 h after which the optical density at 600 nm was measured. Bacteria were collected by centrifugation for 2 min at 12,000 rpm (4 °C). The supernatant was passed through a 0.22-µm filter and then precipitated with trichloroacetic acid (10% final concentration, v/v) at 4 °C for 4–16 h.

Analysis of Secreted Proteins—The trichloroacetic acid-insoluble fraction from above was collected by centrifugation, washed with ice-cold acetone, and solubilized with a volume of 2x SDS-sample buffer (100 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 0.002% bromphenol blue, and 200 mM dithiothreitol) adjusted according to the A₆₀₀ of the original culture. When necessary, solubilized secreted proteins were neutralized with an appropriate volume of non-titrated Tris. The bacterial pellet fraction from above was also dissolved in a volume of 2x SDS-sample buffer adjusted according to the A₆₀₀ of the original culture. Proteins from equivalent numbers of bacterial cells, as determined by A₆₀₀ readings, were separated on 10% or 12% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and then blocked in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST) and 5% (w/v) powdered non-fat milk for 1 h at room temperature. Blots were incubated with the following primary antibodies in TBST plus 5% nonfat milk:rabbit affinity-purified antibodies raised against recombinant SseB, SseD, SseG, and SseE (1:1500), mouse anti-HA monoclonal antibody (1:2000; Covance), and mouse anti-DnaK monoclonal antibody (1:3500; Stressgen). Secondary antibodies conjugated to horseradish peroxidase were used at a 1:5000 dilution in TBST for 1 h at room temperature. Antibody complexes were detected using enhanced chemiluminescence (Amersham Biosciences).

Chemiluminescent -Galactosidase Assays—sseA promoter activity was examined using transcriptional fusions to lacZ and a chemiluminescence assay described by Kehres et al. (32). Approximately 1 kb of chromosomal DNA upstream of the sseA start codon was transcriptionally fused to the lacZ gene and was used to generate a single copy chromosomal fusion, P_sseA::lacZ, in wild-type Salmonella and in various SPI-2 mutants by homologous recombination as described above. Similar lacZ reporter strains were constructed but terminated at different regions of the sseA operon, including sseD and sseE, to generate P_sseD::lacZ and P_sseE::lacZ. LacZ reporter strains were cultured overnight in LB medium, then washed twice in LPM medium and inoculated into fresh LPM medium at either pH 7.0 or 5.8 to give an optical density reading of 0.05 at 600 nm. Cultures were incubated with shaking at 37 °C for 3–8 h, and samples were removed every hour for enumeration of colony forming units and for -galactosidase activity assays. For -galactosidase assays, 0.2 ml of the culture was removed and the bacteria were pelleted by centrifugation. The supernatant was discarded and the bacterial pellet was stored at –20 °C until the end of the experiment. Each pellet fraction was resuspended in 0.2 ml of phosphate-buffered saline, and then 50 µl of chloroform was added to lyse the bacteria. 2 µl of the lysate was transferred to a well of a black microtiter plate containing 100 µl of Galacto-Star substrate mix (Applied Biosystems, Bedford, MA). Reactions were incubated for 60 min at room temperature, and then the plates were read by using the luminescence detection function of the Spectrafluor Plus (TECAN, Austria). Light emission was expressed as relative light units per 10⁶ bacteria based on cfu determinations derived from matched bacterial cultures. Each condition was performed in quadruplicate and averaged.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Salmonella Requires Low pH and an Intact SPI-2 Type III Secretion Apparatus for Secretion of Filament and Translocon Components—To examine the molecular basis of SPI-2 gene expression we performed in vitro secretion assays to study the SPI-2 translocon. We first established and optimized in vitro conditions for SPI-2 secretion assays using minimal medium previously reported to induce the expression of SPI-2 genes (8) and affinity-purified antibodies against SseB and SseD. Using this optimized procedure we were able to reproducibly isolate SPI-2-secreted protein from 1.5 ml of non-stationary culture without the need to extract the bacterial surface with hexadecane (22) or mechanical shearing (8), which had previously been required to detect sufficient levels of SPI-2 translocon components. Wild-type Salmonella and a Salmonella invA::Kan mutant that has a generalized defect in SPI-1-mediated type III secretion were then tested for the ability to secrete the filament component, SseB, and the translocon component, SseD, when the bacteria were grown in low phosphate, low magnesium-containing medium (LPM) at pH 7.0 or 5.8. In secreted protein fractions, SseB and SseD were detected from wild-type and SPI-1 mutant bacterial cultures grown only in LPM medium at pH 5.8 and not in LPM medium at pH 7.0 (Fig. 1A). SseB and SseD secretion required SsaR, a conserved component of type III secretion systems, because an ssaR mutant did not secrete these molecules under these same conditions. To control for the presence of cytosolic proteins in secreted protein fractions due to bacterial lysis, we probed each fraction with an antibody against the cytoplasmic molecule, DnaK, which was negative in all cases.

View larger version (27K): [in this window] [in a new window]   FIG. 1. Expression and SPI-2 secretion of translocators is acid-inducible and requires SsaL and a functional SPI-2 type III secretion system. A, analysis of SPI-2-secreted proteins. Bacterial cultures were grown in low phosphate, low magnesium (LPM) medium at neutral or acidic pH and SseB (top panel) and SseD (middle panel) were detected in the secreted protein fraction by Western blot analysis. Secreted protein fractions were also probed for an intracellular marker, DnaK (bottom panel). B, detection of SseB and SseD in Salmonella lysates. Whole bacterial cell lysates from the cultures from A were subjected to Western blot analysis for SseB and SseD. Lysates were also probed for DnaK (bottom panel). C, detection of SseB from stationary phase Salmonella cultures. Bacterial cultures were grown for 16 h to stationary phase in the indicated media and subjected to Western blot analysis for SseB in whole cell lysates. Expression and secretion assays were repeated five times with similar results. All proteins fractions were normalized to A600 readings for each culture, and differences between the growth of cultures in acidic or neutral minimal medium were not detected (D).

View larger version (28K): [in this window] [in a new window]   FIG. 2. Complementation studies of ssaL. A, effect of overexpression of SsrA/B. ssaL Salmonella containing the plasmid, pssrAB were grown in LPM medium (pH 5.8), and proteins from equal numbers of cfu were subjected to Western analysis for SseB (top panel), SseD (middle panel), and DnaK (bottom panel). B, complementation of SseB expression by re-introduction of a wild-type ssaL allele. Wild-type Salmonella and a ssaL strain each containing pssaL were grown in LPM medium (pH 5.8) and protein from equal numbers of bacteria were subjected to Western analysis for SseB in the whole bacterial fraction (pellet) and the secreted protein fraction. As a control, protein samples were probed for the bacterial cytoplasmic molecule, DnaK. C, the SsaL homologue, SepL from EHEC, moderately restores expression of SseB in whole bacterial lysates (pellet) but does not restore secretion of SseB. The sepL gene from EHEC was expressed in wild-type Salmonella and the ssaL mutant by growing cultures in LPM medium at pH 7.0 or 5.8. SseB in whole bacterial lysates and in secreted proteins fractions was detected by Western blot.

The SepL Homologue, SsaL, Is Required for Translocator Secretion and Expression—Our laboratory has shown that the LEE-encoded secretion regulator SepL is required for secretion of the translocon components EspA, EspB, and EspD, but not for secretion of the effector molecule, Tir, in attaching and effacing pathogens (20). sepL has a single homologue in sequence databases, called ssaL, which is an uncharacterized open reading frame within SPI-2. To test whether SsaL is required for secretion of SPI-2 translocators, we constructed an in-frame, unmarked deletion in the ssaL gene of SPI-2 and performed expression and secretion assays for SseB and SseD. SsaL was required for secretion (see Fig. 3) and full expression (Fig. 1B) of both SseB and SseD. Together, these results demonstrate that a functional SPI-2 TTSS, including SsaL, is required for the expression of the SPI-2 translocon.

View larger version (22K): [in this window] [in a new window]   FIG. 3. SseB is stable in the cytoplasm in the absence of SPI-2 type III secretion. Wild-type Salmonella and the ssaL and ssaR strains expressing SseA, -B, -C, and -D from the constitutive lac promoter (psseA-Dc) were grown in LPM medium at neutral and acidic pH. Each strain containing an empty plasmid (pWSK129) was also cultured under identical conditions. SseB was detected in the secreted protein fraction (A) and the whole bacterial lysate (B) from each of the strains by Western blotting. Lanes contain proteins from equal numbers of bacteria as determined by A600 readings.

Cytoplasmic SseB Is Not Labile in the Absence of SPI-2-dependent Type III Secretion—One possible explanation for the lack of SseB and SseD in Salmonella cells grown in LPM medium at pH 7.0, and in ssaL and ssaR mutants at pH 5.8 is that these type III secretion substrates could be degraded in the absence of secretion. To test this possibility, we constructed Salmonella strains that expressed SseB along with its chaperone, SseA (33, 34), from a constitutive promoter and then tested these strains for SseB using expression and secretion assays. As shown in Fig. 3A, Salmonella strains containing either an empty plasmid or the sseB expression construct did not secrete SseB in LPM at pH 7.0 as expected from the above experiments. However, when these same strains expressed SseB from the constitutive lac promoter, SseB was detected in the secreted protein fraction from wild-type Salmonella grown in LPM medium at pH 5.8 but not from the ssaR or ssaL mutants (Fig. 3A), thereby confirming that both SsaR and SsaL are required for translocator secretion. We also tested whole bacterial lysates from the same strains for the presence of SseB. As shown in Fig. 3B, SseB accumulated in bacteria grown in LPM medium at pH 7.0 when SseB was expressed from the constitutive lac promoter, indicating that in the absence of SPI-2-dependent type III secretion, intracellular SseB is not degraded in the bacterial cytosol. In LPM medium at pH 5.8, both the ssaL and ssaR strains expressing constitutive SseB also accumulated non-secreted, intracellular pools of SseB with no visible breakdown products (Fig. 3B). These data demonstrate that intracellular SseB is not inherently labile in the absence of SPI-2-dependent type III secretion and is stably maintained in the cytoplasm of type III secretion mutants and when Salmonella is subjected to environmental conditions that restrict SPI-2 secretion activity. It was also noted that in wild-type Salmonella, which overexpressed SseA, -B, -C, and -D, the levels of SseB secreted into the culture supernatant was lower than for non-transformed wild-type cells (Fig. 3A). It is possible that this resulted from the increased competition for the SPI-2 apparatus by three overexpressed type III secretion substrates (SseBCD), or possibly due to titration by overexpressed SseC or SseD of an essential endogenous chaperone required for SseB secretion.

The SPI-2 Regulator SsaL Is Required for Salmonella Virulence in Vitro and in Vivo—To validate the significance of SsaL for Salmonella disease, we tested the ability of the ssaL mutant strain to replicate in macrophages and for virulence in vivo. The ssaL strain did not replicate in the murine macrophage cell line RAW 264.7 (Fig. 4A). The numbers of intracellular wild-type Salmonella increased 3.5-fold over 21 h, whereas the ssaL deletion strain showed a net decrease of intracellular bacteria over the same time period. The numbers of intracellular bacteria at 2 h was similar for both wild-type and the ssaL mutant indicating that invasion was not affected (data not shown). The level of attenuation was similar to that of a Salmonella strain deleted for an essential SPI-2 chaperone (sseA) or a conserved type III apparatus component (ssaR). The ssaL strain was also attenuated for intracellular replication in HeLa cells, which was evident at time points longer than 8-h post-infection (data not shown). The intracellular replication defect in the ssaL strain was due to the loss of ssaL, because complementation of the allele on a plasmid under the control of its native promoter restored intracellular growth (Fig. 4A).

View larger version (14K): [in this window] [in a new window]   FIG. 4. ssaL is required for Salmonella virulence in vitro and in vivo. A, gentamicin protection assays. RAW264.7 murine macrophages were infected with wild-type Salmonella and the SPI-2 mutants shown, and the numbers of intracellular bacteria were enumerated at 2 and 20 h after infection. Data are presented as the mean -fold increase in intracellular bacteria with standard deviation from three separate experiments. B, survival plots for BALB/c mice (n = 5) infected with wild-type S. Typhimurium (black squares), ssaR S. Typhimurium (black diamonds), ssaL S. Typhimurium (black triangles), and the complemented ssaL strain (black circles). Percentages of the mice surviving on each day after infection are shown. Replication and virulence experiments were repeated three times.

SsaL Requires an Acidic Minimal Medium and the SPI-2 Regulator, SsrB, for Expression—To further characterize the expression requirements for SsaL, we constructed a plasmid that expressed SsaL with a C-terminal HA epitope and expressed this construct in wild-type Salmonella and in ssaR and ssaL mutants. Expression of SsaL did not occur in minimal medium at neutral pH, consistent with the expression patterns observed for other SPI-2-encoded molecules (Fig. 5A). However, upon acidification of the medium, expression of SsaL was induced in wild-type Salmonella and in ssaR and ssaL mutants (Fig. 5A), indicating that SPI-2 type III secretion activity is not required for SsaL production. SsaL-HA was also not a substrate for SPI-2-dependent type III secretion (Fig. 5A). To test whether expression of the ssaL gene is regulated by the SsrA/SsrB system in response to low pH, we expressed SsaL-HA in a Salmonella mutant with an inactive allele of ssrB. Expression of SsaL was completely abolished in the absence of SsrB (Fig. 5B), indicating that the role for SsaL in expression/secretion of the filament and pore-forming components of SPI-2 is downstream from the activation of SsrA/SsrB.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Expression of ssaL requires acidic pH and the SPI-2 regulator, SsrB. A, Salmonella strains that expressed SsaL-HA or that were transformed with empty plasmid were grown in LPM medium at pH 7.0 and 5.8, and cell fractions were probed for the presence of SsaL-HA. SsaL-HA was not detected in LPM (pH 7.0) medium in any of the strains tested, but was induced upon acidification of the minimal medium (upper panel). SsaL-HA was not detected in the secreted fraction from any of the strains tested (lower panel). B, expression of ssaL is SsrB-dependent. A Salmonella mutant with an inactive allele of ssrB was tested for expression of epitope-tagged ssaL. Whereas wild-type Salmonella expressed SsaL-HA under acidic conditions, SsaL-HA expression was abolished in the absence of SsrB.

View larger version (29K): [in this window] [in a new window]   FIG. 6. SPI-2-encoded effectors are expressed in acidic minimal medium and require SsaL for secretion but secretion of non-SPI-2-encoded effectors is retained. Wild-type Salmonella and ssaL, ssaR, and invA mutants were grown in LPM medium at neutral and acidic pH and tested for presence of SseG and SseE in whole bacterial lysates (A) and in secreted protein fractions (B). As a control, each fraction was also probed for DnaK. SPI-2 effectors encoded outside of the SPI-2 pathogenicity island, including PipB (C), were also examined for their expression and secretion from various Salmonella strains. D, expression of PipB does not restore the expression of SseB or SseD in whole bacterial lysates.

View larger version (16K): [in this window] [in a new window]   FIG. 7. Activation of the sseA promoter requires acidic minimal medium and a functional SPI-2 type III secretion system. A, wild-type Salmonella with a single chromosomal integration of a PsseA::lacZ reporter fusion was grown in LPM medium at pH 7.0 and 5.8 for the times indicated. -Galactosidase activity and bacterial counts were measured at each time point, and data are expressed as the relative light units of -galactosidase activity per 1 x 106 cfu. Data are the means with standard errors from triplicate determinations. B, SL1344 PsseA::lacZ (closed squares), ssaR PsseA::lacZ (open circles), and ssaL PsseA::lacZ (closed diamonds) strains were grown in LPM medium at pH 5.8, and -galactosidase activity was measured at the indicated time points. Data are the means with standard errors from quadruplicate determinations. *, p < 0.01 compared with wild-type strain. C, Salmonella reporter strains were cultured in LPM medium at neutral pH, and -galactosidase activity was measured as described above. Data are the means with standard errors from quadruplicate determinations, and the experiments were repeated at least three times.

Transcriptional Activity of Genes Downstream of the Poreforming Translocon Genes Can Be Uncoupled from the Requirement of Type III Secretion and SsaL—The expression experiments shown in Fig. 6 demonstrated that, although the expression of effectors downstream of the filament and poreforming genes (SseB and SseD) was also pH-dependent, the ssaR and ssaL strains expressed similar amounts of SseE and SseG compared with wild-type Salmonella, which contrasts with the expression pattern for the upstream genes. To examine this, we constructed single-copy chromosomal transcriptional fusions of lacZ to the sseD gene and the immediately downstream gene, sseE, and measured -galactosidase activity from wild-type, ssaR and ssaL reporter strains. SseD is the second component of the pore-forming translocon (8). As shown in Fig. 8, -galactosidase activity from Salmonella strains with the P_sseD::lacZ reporter was comparable to the activity for the P_sseA::lacZ fusion in terms of magnitude and demonstrated lower activity in the ssaR and ssaL mutants compared with wild-type. -Galactosidase activity from the P_sseE::lacZ reporter were not significantly different between wild-type Salmonella or the ssaR and ssaL mutants, thus confirming the expression patterns observed previously for SseE. -Galactosidase activity from the P_sseE::lacZ reporter strains was 3-fold higher than either the P_sseD::lacZ or P_sseA::lacZ reporters in all strains at all time points tested (Fig. 8). Together with the protein expression data, these data suggest that expression of the genes downstream of the filament (sseB) and pore-forming component of the SPI-2 translocon (sseD) can be uncoupled from the expression of the upstream genes with respect to the requirement of type III secretion and the presence of SsaL.

View larger version (22K): [in this window] [in a new window]   FIG. 8. Differential regulation and activity of sseD and sseE::lacZ reporters. Single-copy chromosomal integrations of the reporters, PsseD::lacZ (solid lines) and PsseE::lacZ (dashed lines) were constructed in wild-type Salmonella (SL1344) and in the ssaR and ssaL mutants. -Galactosidase activity was measured at the indicated time points from whole bacterial lysates and expressed as relative light units of -galactosidase activity per 1 x 106 cfu. Data are the means with standard errors from quadruplicate determinations. p < 0.05 for wt PsseD::lacZ compared with ssaR PsseD::lacZ or ssaL P::lacZ; psseD < 0.01 for wt PsseD::lacZ compared with wt PsseE::lacZ.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It is known that the two-component regulatory system comprised of SsrA/SsrB positively regulates SPI-2-encoded genes. SsrA is the putative sensor kinase component and SsrB is the transcriptional activator that acts on promoters in SPI-2 and in other regions of the genome. Using transcriptional fusions to green fluorescent protein, Lee and colleagues (15) demonstrated that the promoter controlling ssrAB was activated in acidic minimal medium but not in minimal medium at neutral pH. We therefore reasoned that genes controlled by SsrA/SsrB (such as the sseA operon) should also demonstrate acid-induced activation. We found that components of the SPI-2 filament (SseB), translocon (SseD), and effectors (SseG and SseE) required acidified minimal medium for expression and accumulation within wild-type bacteria. It was of interest to find that SseE was localized to bacterial cells but was not found in the secreted fraction from any of the Salmonella strains tested. Although there are currently no published data on SseE, based on its localization within Salmonella cells, we hypothesize that it could play a chaperone role or other accessory role for SPI-2-dependent type III secretion.

As mentioned, a previous report demonstrated that activation of the ssrA promoter required minimal medium at acidic pH (15). However, two other studies investigating SPI-2 gene expression (22, 23) reported that the expression of SPI-2 genes was pH-independent. These data are difficult to reconcile with each other given that SsrA/SsrB is a requisite activator of SPI-2 gene expression. Our results are in agreement with the former study (15) and show that activation of the sseA promoter and expression of both translocon components (SseB and SseD) and effectors (SseG and SseE) requires acidic pH. This pH-inducible activation was SsrB-dependent, because no SPI-2 protein accumulation or secretion was seen in an ssrB mutant. Given that the SCV acidifies to below pH 5.5 within 20 min after uptake into bone marrow-derived macrophages (24), it's likely that an acidic pH is a major environmental cue determining not only activation of SPI-2 secretion, but also inducing the expression of SPI-2-encoded genes. We suspect that the bacterial growth stage represents an important contribution to the regulation of SPI-2, because we found that growth of Salmonella to stationary phase in neutral minimal medium, conditions that were used in previous studies that did not find pH-dependent SPI-2 gene expression, caused accumulation of SseB in wild-type Salmonella but not in ssaL or ssaR mutants. Whether this represents a stress response by the bacteria will require additional experimentation. It should be noted that, in our expression and secretion experiments, all bacterial cultures were tested during exponential-phase growth, after 4–6 h of incubation in minimal medium. The sensitivity of our expression and secretion assays allowed us to perform these experiments before the bacterial cultures reached saturation in stationary phase and allowed us to detect SPI-2-secreted proteins from culture volumes of <2 ml without the need to extract the bacterial surface by chemical or mechanical means, conditions that were previously reported as being necessary to detect SPI-2-secreted proteins in vitro (8, 22).

The data presented here are consistent with the hypothesis that the pH-dependent secretion of a putative transcriptional repressor may play a role in controlling SPI-2 gene expression and that SsaL might be involved in this process. In such a model, repression of the translocon promoter upstream of sseA would occur in the absence of SPI-2 type III secretion, which is inhibited during bacterial growth in neutral minimal medium or disabled in SPI-2 apparatus mutants. Following activation of SPI-2-dependent type III secretion, as when bacteria encounter an acidified intracellular environment, the active secretion of a putative repressor would lower its cytoplasmic concentration and de-repress the translocon promoter, facilitating SsrB-mediated expression of SseB, -C, and -D. Given that type III secretion systems have shown a hierarchal secretion of regulators, translocators, and effectors (37), it is possible that an SPI-2 TTSS intermediate exists in which the apparatus is permissive for secretion of regulatory controllers prior to engaging translocators and effector substrates. This transitional structure would represent a secretion-competent but not translocation-competent intermediate in which substrates could be secreted into the SCV lumen prior to formation of the filament and translocon pore. Given that ssaL mutants constrain the expression of the SPI-2 translocon, it is possible that SsaL is involved in such a transitional secretion complex. This hypothesis is supported by data from Cirillo and colleagues (38), who showed that a Salmonella sseB mutant (that can still secrete, but not translocate, SPI-2 substrates) could still activate promoters controlling the SPI-2 molecules, SpiA (SsaC), SscB, and SsrA. This group also reported that SPI-2 gene expression did not require an intact secretion apparatus. However, this conclusion may be limited insofar as the expression studies were performed with a limited number of SPI-2 mutants containing plasmid-based transcriptional fusions to GFP, whereas it has been observed that SPI-2 gene expression can be improperly regulated when SPI-2 genes are expressed from medium copy plasmids from their native promoters (9).² It is possible that an increased copy number of SPI-2 promoters in these instances could titrate out a putative repressor molecule leading to the observed gene expression patterns from plasmid-based studies. Although it is possible that SsaL is part of the core type III apparatus, genetic and phenotypic evidence might suggest a more complex role. First, SsaL has only one homologue (SepL) in type III secretion systems from other pathogens, whereas it has been recognized that the core structure of the type III apparatus is generally well conserved (21, 39). Second, SepL from attaching and effacing gastrointestinal pathogens has been shown to regulate the transition from translocon to effector secretion (20) and is dispensable for secretion of type III effectors. Third, SepL from EHEC can moderately complement an ssaL mutation in Salmonella for expression of SseB, but not for secretion, suggesting a bifunctional role for SsaL in SPI-2, and lastly, an ssaL mutant retains the ability to secrete non-SPI-2-encoded effectors, whereas a generalized secretion mutant (ssaR) does not.

Interestingly, Day and Lee (40) recently hypothesized that an SPI-1-encoded protein called OrgC functions as a SPI-1-secreted repressor of SPI-1 virulence genes. This hypothesis was based on phenotypic studies using an orgC deletion mutant and by comparing the gene synteny of orgC to that of virulence genes in other pathogens. In particular, the position of orgC in the SPI-1 operon, prgHIJKorgABC corresponds to the position of a transcriptional repressor, lcrQ in the Yersinia virulence plasmid (40). Although the hypothesis that OrgC is a transcriptional repressor of SPI-1 was not formally tested by Day and Lee, it remains plausible that a corresponding mechanism exists to repress SPI-2 gene expression until the appropriate intracellular environmental cue(s) is sensed.

It was of interest that SPI-2-encoded effectors and translocators were repressed in a similar fashion in minimal medium at neutral pH, but there was no difference in SseE or SseG expression between wild-type, ssaR, and ssaL mutants in acidic minimal medium. It is possible that SPI-2-encoded effectors downstream of sseD are subject to different regulatory control compared with the translocators, despite being present in the same putative operon. Transcription fusions of lacZ to various regions of this operon, including sseD and the immediately downstream gene, sseE, confirmed that the expression of upstream translocators and downstream "effectors" has different requirements with respect to type III secretion and the presence of SsaL. These data also suggest the presence of a previously unrecognized regulatory element that might act specifically on genes downstream of the translocon, because the activity of the P_sseE::lacZ reporter was significantly greater than two upstream reporters. Indeed, there is already evidence that SPI-2 genes in the same transcriptional unit contain distinct promoters that are differentially activated. For example, Feng and colleagues (41) recently reported that, unlike many two-component regulatory systems, regulation of the sensor kinase SsrA is partially uncoupled from regulation of the response regulator SsrB in SPI-2, owing to their distinct promoters. Emerging evidence implies that combinatorial pair-wise interactions of different response regulators can interact at the same promoter, suggesting an increase in the complexity of prokaryotic transcriptional regulation akin to that seen in eukaryotic systems (42–44). Because SPI-2 gene regulation is integral to Salmonella pathogenesis, it is reasonable to speculate that virulence gene regulation in Salmonella is under complex regulatory control, and insight into this process will lead to gains in understanding how intracellular Salmonella interact with host cells to cause disease.

	FOOTNOTES

 
^* This work was supported in part by grants (to B. B. F.) from the Canadian Institutes of Health Research (CIHR) and the Howard Hughes Medical Institute (HHMI). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a CIHR postdoctoral fellowship and a Michael Smith Foundation for Health Research fellowship.

^¶ Recipient of a CIHR New Investigator Award. Current address: Infection, Immunity, Injury and Repair Program, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.

A CIHR Distinguished Investigator, an HHMI International Research Scholar, and the University of British Columbia Peter Wall Distinguished Professor. To whom correspondence should be addressed: Biotechnology Laboratory, 237-6174 University Boulevard, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3. Tel.: 604-822-2210; Fax: 604-822-9830; E-mail: bfinlay{at}interchange.ubc.ca.

¹ The abbreviations used are: SPI, Salmonella pathogenicity island; TTSS, type III secretion system; SCV, Salmonella-containing vacuole; LEE, locus of enterocyte effacement; EHEC, enterohemorrhagic E. coli; EPEC, enteropathogenic E. coli; cfu, colony-forming unit(s); LPM, low magnesium, low phosphate-containing medium; HA, hemagglutinin; MES, 2-(N-morpholino)ethanesulfonic acid.

² B. Coombes and B. Finlay, unpublished data.

	ACKNOWLEDGMENTS

 
We thank M. Wickham, J. Puente, P. Hardwidge, and G. Grassl for helpful discussions and insightful comments. We also acknowledge W. Deng for sharing unpublished data on Citrobacter rodentium sepL, G. Gotto for assistance with antibody production, and M. Hensel for providing pssrAB.

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