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Infection and Immunity, January 2005, p. 334-341, Vol. 73, No. 1
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.1.334-341.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Salmonella Pathogenicity Island 2-Mediated Overexpression of Chimeric SspH2 Proteins for Simultaneous Induction of Antigen-Specific CD4 and CD8 T Cells

Klaus Panthel,1 Katrin M. Meinel,1 Victòria E. Sevil Domènech,1 Heike Retzbach,1 Emeka I. Igwe,1 Wolf-Dietrich Hardt,2 and Holger Rüssmann1*

Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig Maximilians Universität München, Munich, Germany,1 Institut für Mikrobiologie, ETH Zürich, Zürich, Switzerland2

Received 29 July 2004/ Returned for modification 8 September 2004/ Accepted 14 September 2004


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ABSTRACT
 
Salmonella enterica serovar Typhimurium employs two different type III secretion systems (TTSS) encoded within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) for targeting of effector proteins into the cytosol of eukaryotic cells during different stages of the infection cycle. The SPI1 TTSS translocates virulence factors across the plasma membrane when the bacterium initially contacts the host cell. In contrast, the SPI2 TTSS functions to translocate proteins across the membrane of the Salmonella-containing vacuole and promotes intracellular survival and replication. The aim of the present study was to directly compare the potentials of SPI1 and SPI2 type III effector proteins to act as carrier molecules for a heterologous antigen. The p60 protein of Listeria monocytogenes was used as a model antigen to construct chimeric SopE2 (SPI1), SifA (SPI2), and SspH2 (SPI2) proteins. SPI1- and SPI2-dependent up- and down-regulation of hybrid gene expression led to sequential translocation of p60 fusion proteins into the cytosol of Salmonella-infected macrophages. Mice orally immunized with recombinant Salmonella strains expressing these hybrid proteins revealed comparable numbers of p60-specific CD8 T cells. However, only overexpression of translocated SspH2/p60 from a medium-copy-number vector induced simultaneous antigen-specific CD4 and CD8 T-cell responses, suggesting that SspH2 is an attractive carrier molecule for foreign-protein delivery.


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INTRODUCTION
 
In Salmonella enterica serovar Typhimurium, two virulence-associated type III secretion systems (TTSS) are encoded by separate pathogenicity islands, Salmonella pathogenicity island 1 (SPI1) and SPI2 (15, 22, 24, 46). Upon close contact with the eukaryotic target cell, the TTSS encoded by SPI1 mediates Salmonella invasion of the host cell, where the bacterium resides within a membrane-bound compartment called a Salmonella-containing vacuole (SCV), or macropinosome (1, 36). In contrast, the TTSS encoded by SPI2 is activated under intracellular conditions and is required for the intracellular survival and proliferation of the bacterium (22). By means of these specialized secretion systems, Salmonella translocates type III effector proteins into the host cell cytoplasm either (i) from extracellular sites and from the SCV at an early stage of invasion (SPI1 mediated) (10, 15) or (ii) from the SCV at a significantly later stage during intracellular survival and replication (SPI2 mediated) (8, 21).

Attenuated Salmonella carrier vaccines have the potential to be used as delivery systems for foreign antigens from pathogens of viral, bacterial, and parasitic origin (11). It is well known that Salmonella residing within the SCV is not an ideal vector to deliver heterologous proteins to the cytosol of host cells, which would lead to major histocompatibility complex (MHC) class I-restricted antigen presentation (42, 43). In fact, secretion of foreign proteins into the macropinosomal compartment by Salmonella results in MHC class II-restricted antigen presentation and peptide-specific CD4 T-cell priming (52). Our laboratory has focused its research on the genetic manipulation of attenuated Salmonella strains to endow them with the ability for efficient induction of MHC class I-restricted immune responses (25, 42, 43, 44). We have developed a new vaccination strategy by using the SPI1-TTSS to translocate antigens from Listeria monocytogenes directly into the cytosol of antigen-presenting cells (APC). The immunodominant listerial proteins p60 and listeriolysin O (LLO) were fused to the defined N-terminal translocation domain of the Yersinia outer protein E (YopE). In vitro experiments revealed that Salmonella allows secretion and translocation of these chimeric proteins in a TTSS-dependent fashion (43). Translocation and cytosolic delivery of hybrid proteins into host cells, but not secretion into endosomal macropinosomes, led to efficient MHC class I-restricted antigen presentation of listerial nonamer peptides. As determined by enzyme-linked immunospot (ELISPOT) assays, mice orally vaccinated with a single dose of attenuated Salmonella expressing either translocated YopE/LLO or YopE/p60 proteins revealed high numbers of gamma interferon (IFN-{gamma})-producing CD8 T cells reactive with LLO91-99 or p60217-225, respectively. These T lymphocytes conferred protection against a lethal challenge with a Listeria wild-type strain (43).

The goal of this study was to directly compare the potentials of SPI1 and SPI2 type III effector molecules that are known to be expressed at different stages of the Salmonella infection cycle to act as carrier molecules for a heterologous protein containing immunodominant MHC class I- and II-restricted peptides. The p60 protein of Listeria fused to YopE, SopE2, SifA, or SspH2 served as a model antigen. Promoter-dependent up- and down-regulation of hybrid gene expression led to sequential translocation of chimeric p60 proteins into the cytosol of Salmonella-infected macrophages. Mice orally immunized with Salmonella strains translocating p60 via SPI1 or SPI2 revealed comparable numbers of p60-specific CD8 T cells but not a measurable antigen-specific CD4 T-cell response. However, overexpression of two different SspH2/p60 hybrid proteins from a medium-copy-number vector led to concomitant p60-specific CD4 and CD8 T-cell priming, indicating that the SPI2 effector protein SspH2 might be an attractive carrier molecule for antigen delivery when T-cell immune responses against complex microbes or tumors are needed.


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MATERIALS AND METHODS
 
Plasmids, bacterial strains, and growth conditions. Escherichia coli {chi}6060 was used as an intermediate host for cloning procedures. All hybrid proteins used in this study were M45 epitope tagged at their C termini. This tag (MDRSRDRLPPFETETRIL) is derived from the E4-6/7 protein of adenovirus (35), and its use for chimeric-protein tagging has been described (43). The construction of plasmid pHR241 (Table 1) has been outlined in detail (43). This derivative of pWSK29 (51) is a low-copy-number expression vector and bears the genetic information for the translocated chimeric YopE1-138/p60130-477/M45 fusion protein under expression control of the lac promoter, which is constitutively active in serovar Typhimurium.


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  		TABLE 1. Serovar Typhimurium strains and plasmids used in this study

Plasmid pM226, a pBAD24 derivative, is a medium-copy-number expression vector for the M45 epitope-tagged version of SopE21-240 under the control of the native promoter encoded by 833 bp upstream of the sopE2 gene (48). In the next cloning step, a DNA fragment of the iap gene (coding for p60130-477) was amplified using the forward primer p60-1 (5'-GGTCCCGGGAAATACTTAACTGAC-3') and the reverse primer p60-2 (5'-TACCCATGGCAGAGCCGTGGATGT-3'). The PCR product was cloned into the SmaI/NcoI sites of pM226. The resulting plasmid, pHR261, encodes SopE21-240/p60130-477/M45. To construct a pWSK29 derivative without the lac promoter, pWSK29 was digested with BamHI and BglII, and the 1.9-kb DNA fragment missing the promoter was cloned into pWSK29 digested with BglII to obtain pWSK29-w/oP. In a further cloning procedure, pHR261 was digested with NruI and XbaI to retrieve the coding DNA for hybrid SopE2, klenowed, and cloned into the EcoRV site of pWSK29-w/oP. The resulting low-copy-number plasmid, pHR262, encodes SopE21-240/p60130-477/M45 under the control of the native promoter.

The sifA region including the putative promoter encoded by 326 bp upstream of the sifA gene and the entire sifA open reading frame (ORF) was amplified by PCR using the primers sifA-forward (5'-CGGAATTCTGCGCAACGCTAACAAATC-3') and sifA-reverse (5'-TCCCCCGGGTAAAAAACAACATAAACAGCCGC-3'). The latter primer was designed to replace the stop codon of sifA with a SmaI restriction site to allow the construction of fusion proteins. The PCR product was cloned into pCR-Blunt II-TOPO, yielding pM311, and the insert was verified by sequencing. The insert of pM311 harboring the sifA promoter and the ORF was retrieved with SmaI and EcoRI and cloned into the EcoRI/SmaI sites of pSB1136, a pBAD24 derivative (W.-D. Hardt and J. E. Galán, unpublished data), yielding pM313. pM313 carries a C-terminally M45-tagged version of sifA under the control of its native promoter. The above-mentioned forward primer p60-1 and the reverse primer p60-2 were used to amplify a DNA fragment of the iap gene (coding for p60130-477). The PCR product was cloned into the SmaI/NcoI sites of pM313, resulting in pHR271. This medium-copy-number vector bears the genetic information for SifA1-336/p60130-477/M45. In the next cloning step, pHR271 was digested with EcoRI and SalI to retrieve the coding DNA for hybrid SifA and cloned into the EcoRI/SalI sites of pWSK29-w/oP. The resulting low-copy-number plasmid pHR272 encodes SifA1-336/p60130-477/M45 under the control of the native promoter. In a further cloning procedure, a pBAD24 derivative was constructed for the expression of SifA1-336/p60130-477/M45 under the control of the sspH2 promoter. Therefore, a DNA fragment encoding this chimeric protein (forward primer, sifA-A EcoRI [5'-TATGAATTCATTTTTACTCCAGTA-3']; reverse primer, sifA-B SalI [5'-CAGGTCGACTCTAGAGGATCCGCG-3']; template DNA, pHR271) was amplified and cloned into the EcoRI/SalI sites of pM313 to obtain pHR496. The sspH2 promoter region (template pM314; see below) was amplified using the forward primer sspH2-P-1 EcoRI (5'-AGAGAATTCACTAGTTGCCTGATACGG-3') and the reverse primer sspH2-P-2 EcoRI (5'-CCGGAATTCAACAAAAAACCTTTATAAATT-3'). The PCR product was cloned into the EcoRI site of pHR496, yielding pHR273.

The sspH2 region including the putative promoter encoded by 361 bp upstream of the sspH2 gene and the 5'-terminal 642 nucleotides of the sspH2 ORF was amplified by PCR using the primers sspH2-forward (5'-GACTAGTTGCCTGATACGGATGAAAACC-3') and sspH2-reverse (5'-ATCGGAAGACCTGTTCTCCCACG-3'). The latter primer was designed to introduce a SmaI restriction site at the 3' end of the truncated sspH2 ORF to allow the construction of fusion proteins. The PCR product was cloned into pCR-Blunt II-TOPO, yielding pM310, and the insert was verified by sequencing. The insert of pM310 harboring the sspH2 promoter and the truncated ORF was retrieved by digestion with PstI, Klenow treatment, and digestion with SpeI and was cloned into the SmaI/SpeI sites of pM226 (a pBAD24 derivative) (48), yielding pM314. pM314 carries a C-terminally truncated and M45-tagged version of sspH2 under the control of its native promoter. In the next cloning step, a DNA fragment of the iap gene (coding for p60130-477) was amplified using the forward primer p60-3 (5'-ACTGAATTCGTTAACGGTAAATAC-3') and the above-mentioned reverse primer p60-2. The PCR product was cloned into the EcoRI/NcoI sites of pM314. The resulting plasmid, pHR281, encodes SspH21-214/p60130-477/M45. This vector was digested with EcoRV and SalI to retrieve the coding DNA for hybrid SspH2 and cloned into the EcoRV/SalI sites of pWSK29-w/oP. The resulting low-copy-number plasmid, pHR282, encodes SspH21-214/p60130-477/M45 under the control of the native promoter. In a further cloning procedure, a pBAD24 derivative was constructed for the expression of SspH21-214/p60130-477/M45 under the control of the sifA promoter. Therefore, two separate DNA fragments were amplified: an upstream fragment containing the sifA promoter region (forward primer, sifA-P EcoRI [5'-GAGGAATTCTGCGCAACGCTAACA-3']; reverse primer, sifA-P SmaI [5'-AGTCCCGGGCATATTAATCTCACTTAT-3']; template DNA, pM313) and a downstream fragment harboring the genetic information for SspH21-214/p60130-477/M45 (forward primer, sspH2-A SmaI [5'-GTTATGCCCGGGCATATTGGAAGCGGA-3']; reverse primer, sspH2-B SalI [5'-CAGGTCGACTCTAGAGGATCCGCG-3']; template DNA, pHR281). The fragments were assembled as cassettes with the EcoRI/SmaI/SalI sites and inserted between the EcoRI/SalI sites of pM313 to yield pHR283. In the next cloning step, a pBAD24 derivative was constructed for the expression of SspH21-214/p60130-477/M45 under the control of the lac promoter. Therefore, two separate DNA fragments were amplified: an upstream fragment containing the lac promoter region (forward primer, lac-P EcoRI [5'-ACAGAATTCCCGACTGGAAAGCGG-3']; reverse primer, lac-P SmaI [5'-TAGCCCGGGCGCCACCGCGGTGGA-3']; template DNA, pWSK29) and a downstream fragment harboring the genetic information for SspH21-214/p60130-477/M45 (forward primer, sspH2-C SmaI [5'-AAGCCCGGGATGTCAGGACGACCA-3']; reverse primer, sspH2-B SalI [see above]; template DNA, pHR281). The fragments were assembled as cassettes with the EcoRI/SmaI/SalI sites and inserted between the EcoRI/SalI sites of pM313 to yield pHR284. In a further cloning procedure, a pBAD24 derivative was constructed for the expression of SspH21-151/p60130-477/M45 under the control of the native sspH2 promoter. Therefore, a DNA fragment coding for this promoter and SspH21-151 was amplified using the forward primer sspH2-D (5'-CCTACCTGACGCTTTTTATCGCAA-3') and the reverse primer sspH2-E EcoRI (5'-CCTGAATTCCCCGGATGCCCCTTC-3') (template DNA pM314) and cloned into the SpeI/EcoRI sites of pHR366 to yield pHR285.

The above-described plasmids were transformed into S. enterica serovar Typhimurium strain SB824 by electroporation (42). Strain SB824 was engineered by introducing the sptP::kan mutant allele from strain SB237 (26) into the {Delta}aroA strain SL3261 (23) by P22HTint transduction. Serovar Typhimurium was grown in Luria-Bertani medium supplemented with 0.3 M NaCl, pH 7.0, to allow the expression of components and targets of the TTSS encoded by SPI1 (9).

Western blot analysis of translocated hybrid type III proteins in Salmonella-infected P388D1 cells. The detection of translocated hybrid type III proteins was carried out as described by Collazo and Galán (10). Briefly, P388D1 cells were grown for 2 days in Dulbecco modified Eagle medium supplemented with 5% fetal bovine serum in 100-mm-diameter tissue culture plates to reach 70% confluency. Next, 1 h before the addition of bacteria, the culture medium was replaced with 500 µl of Hanks' balanced salt solution (HBSS). The bacteria were grown overnight for 12 h in Luria-Bertani medium supplemented with 0.3 M NaCl, diluted 1/20 in fresh medium, and grown for another 4 h under mild aeration to reach an optical density at 600 nm of 0.9. P388D1 cells were infected with serovar Typhimurium for 90 min with a multiplicity of infection of 10 bacteria per cell. After infection, nonadherent bacteria were removed and the cells were washed with HBSS. The infected P388D1 cells were incubated for 30 min with Dulbecco modified Eagle medium containing 100 µg of gentamicin/ml to kill extracellular bacteria. Biochemical fractionation of the cells was performed either 2, 6, or 24 h after infection. P388D1 cells were treated with 30 µg of proteinase K/ml in HBSS for 15 min at 37°C to eliminate cell surface-associated hybrid type III proteins. After the proteinase K treatment, 3 ml of chilled HBSS containing 2 mM phenylmethylsulfonyl fluoride was added. The cells detached during the proteinase K treatment, and they were subsequently collected by low-speed centrifugation (600 x g for 10 min) and lysed in 1 ml of HBSS containing 0.1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride. The cell lysate was transferred to a microcentrifuge tube, treated with DNase and RNase for 15 min at room temperature, and centrifuged at 15,000 x g for 10 min. The pellet was resuspended in phosphate-buffered saline (the Triton X-100-insoluble fraction), the supernatant was filtered through a 0.45-µm-pore-size syringe filter, and proteins were precipitated in the presence of 10% trichloroacetic acid (the Triton X-100-soluble fraction). Samples were separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes as described previously (10). Hybrid type III proteins were detected by immunoblot analysis. Western blots were treated with a monoclonal antibody (MAb) directed against M45 (43), followed by horseradish-labeled anti-mouse antibody. The blots were developed by using a chemiluminescence kit.

Oral immunization of mice with recombinant Salmonella. Female BALB/c mice, 6 to 8 weeks old, were purchased from Harlan-Winkelmann (Borchem, Germany). All mice were kept under specific-pathogen-free conditions (positive-pressure cabinet) and were provided with food and water ad libitum. Groups of five mice were orally immunized with a single dose of 108 CFU of the serovar Typhimurium strain SB824. Six weeks after immunization, the mice were sacrificed and the spleens were used for further ELISPOT analysis. Each experiment was performed at least twice with similar results.

ELISPOT assay. The frequency of T lymphocytes in mice immunized with attenuated serovar Typhimurium was determined with an IFN-{gamma}-specific ELISPOT assay (17, 50). The assays were performed in nitrocellulose-backed 96-well microtiter plates (Nunc, Wiesbaden, Germany) coated with rat anti-mouse IFN-{gamma} MAb (RMMG-1; Biosource, Camarillo, Calif.). Unseparated splenocytes (6 x 105/well) were stimulated for 6 h in round-bottom microtiter plates in the presence of a 10–6 M concentration of the CD8 T-cell epitope p60217-225 or the CD4 T-cell epitope p60367-378. Subsequently, activated cells (4 x 105/well) were transferred to ELISPOT plates and incubated overnight. The ELISPOT plates were developed with biotin-labeled rat anti-mouse IFN-{gamma} MAb (clone XMG1.2; Pharmingen, San Diego, Calif.), horseradish peroxidase-streptavidin conjugate (Dianova, Hamburg, Germany), and aminoethylcarbazole dye solution. The frequency of antigen-specific cells was calculated as the number of spots per splenocyte seeded. The specificity and sensitivity of the ELISPOT assay were controlled with IFN-{gamma}-secreting CD8 T-cell lines specific for p60217-225 and CD4 T-cell lines specific for p60367-378. Recovery of the seeded T cells was >90%.

Statistical analysis. The statistical significance of the results of in vivo experiments was checked with the nonparametric Tukey multiple comparison test at the 0.05 significance level. All tests were performed using WINKS statistical analysis software (Texasoft, Cedar Hill, Tex.).


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RESULTS
 
Construction of chimeric p60 proteins for SPI1- or SPI2-dependent translocation. In recent studies, the p60 protein of L. monocytogenes has been used as a model antigen for the construction of hybrid YopE proteins to be delivered by the Salmonella TTSS (25, 43). After invasion of target cells and escape from the phagosome, Listeria constitutively secretes the murein hydrolase p60 (40). Subsequently, p60 is directed to the MHC class I and class II antigen-processing pathway, leading to presentation of antigen-derived peptides to CD8 and CD4 T cells (18, 39). Analysis of T cells from Listeria-infected BALB/c mice revealed that the immunodominant listerial nonamer peptide p60217-225 is presented to cytotoxic T lymphocytes in the context of the H2-Kd MHC class I molecule (37, 38), whereas residues 367 to 378 of p60 are recognized by a CD4 T-cell clone in the context of the MHC class II molecule (18).

Table 1 shows that the previously described low-copy-number plasmid pHR241 (25, 43) bears the genetic information for a YopE/p60 hybrid protein. The N-terminal 138 amino acids of YopE, containing the secretion and translocation domains (45, 47), were fused to p60130-477, and the resulting chimeric protein was tagged at its C terminus with an adenoviral M45 epitope (35). Constitutive expression of the gene fusion led to the production of a hybrid protein that was shown to be translocated into the cytosol of macrophages by serovar Typhimurium (43). In this study, however, we decided to fuse p60 to translocated Salmonella type III proteins that are engaged by either the SPI1 TTSS or the SPI2 TTSS. This strategy should allow the elucidation of the influence of sequential translocation of chimeric p60 on the induction of p60-specific T-cell responses.

Translocated SopE2 is an SPI1 effector protein that acts as a guanine nucleotide exchange factor for host cellular Rho GTPases (3, 13, 48) and plays a role in the early (extracellular) step of Salmonella invasion of host cells (14, 27). We fused p60130-477 to the full-length SopE2 protein. The resulting SopE21-240/p60130-477/M45 hybrid protein is encoded either on a medium-copy-number vector (pHR261) or on a low-copy-number vector (pHR262) under the control of the native sopE2 promoter (Table 1). In contrast, SifA is engaged by the SPI2 TTSS, leading to translocation of the protein across the membrane of the SCV. SifA is necessary for the formation of Salmonella-induced filaments (Sifs) and maintains the integrity of the vacuolar membrane, probably due to the function of its membrane-anchoring C terminus (5, 6). In this study, p60130-477 was fused to full-length SifA1-336 under the control of its native promoter. Again, a medium-copy-number plasmid (pHR271) and a low-copy-number plasmid (pHR272) were used as expression vectors (Table 1). As a third carrier protein for cytosolic delivery of p60, SspH2 was chosen. SspH2 is a leucine-rich repeat protein that is translocated by the SPI2 TTSS (33) and colocalizes with the polymerizing actin cytoskeleton (34). It has been demonstrated that the amino terminus of SspH2 binds to filamin, suggesting that this interaction is important for the subcellular localization of this type III effector protein (34). Because full-length SspH2 consists of 788 amino acids and the 12 leucine-rich regions are located in a domain spanning amino acids 234 to 444, we decided to fuse truncated SspH21-214 to p60130-477 under the control of the native sspH2 promoter, resulting in the medium-copy-number vector pHR281 or the low-copy-number vector pHR282, respectively (Table 1).

Time-dependent translocation of chimeric p60 proteins into the cytosol of macrophages. We investigated whether infection of macrophage-like P388D1 cells with attenuated serovar Typhimurium SB824 expressing SopE2/p60/M45, SifA/p60/M45, or SspH2/p60/M45 would result in time-dependent expression and translocation of these chimeric proteins. Therefore, 2, 6, and 24 h after infection, biochemical fractionations of P388D1 cells were carried out. Two different fractions were examined by immunoblotting for the presence of hybrid proteins (Fig. 1): (i) a Triton X-100-insoluble fraction containing internalized bacteria (fraction 1) and (ii) a Triton X-100-soluble cell lysate containing cytosolic proteins (fraction 2). As a reference chimeric protein for this experimental setup, we used the previously described YopE/p60/M45 encoded by pHR241 (25, 43). As shown in Fig. 1, YopE/p60/M45 was expressed (fraction 1) and efficiently translocated (fraction 2) at all three time points by intracellular Salmonella.



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  		FIG. 1. Fractionation of P388D1 cells infected with serovar Typhimurium strains expressing plasmid-encoded hybrid p60 proteins. Fractionations were carried out 2, 6, and 24 h after infection. Fraction 1, Triton X-100-insoluble fraction containing internalized bacteria; fraction 2, Triton X-100-soluble P388D1 cell lysate containing translocated proteins. The total protein amounts obtained from both fractions were loaded. Chimeric p60 proteins were detected by protein immunoblotting with a MAb to M45. The copy numbers of the respective plasmids are indicated (MC, medium-copy-number vector; LC, low-copy-number vector). Promoters are written in italics, and the respective carrier proteins fused to p60/M45 are indicated.

SopE2/p60/M45, encoded by pHR261 or pHR262, was expressed and translocated during the first 6 h after infection of macrophages, whereas 24 h postinfection, the fusion protein was no longer detectable (Fig. 1). Thus, expression and SPI1 TTSS-mediated translocation of chimeric SopE2/p60 stopped after Salmonella invasion of host cells. Unlike YopE/p60 and SopE2/p60, SifA/p60 encoded by pHR271 or pHR272 could not be detected 2 h after infection of P388D1 cells with Salmonella (Fig. 1). However, after 6 and 24 h, expression and translocation of SifA/p60 were observed in macrophages infected with SB824 carrying the medium-copy-number vector pHR271. In contrast, P388D1 cells infected with SB824 carrying the low-copy-number vector pHR272 revealed a detectable amount of SifA/p60 only 24 h postinfection. The relatively late production of chimeric SifA reflects the function of SPI2 genes, which are maximally expressed only when bacteria have resided intracellularly for several hours. The same pattern of time-dependent expression and translocation observed for SifA/p60 was detected for SspH2/p60/M45 encoded by pHR281 or pHR282 (Fig. 1).

In all experiments, expression from the medium-copy-number vector resulted in more efficient production and translocation of the respective chimeric protein than expression from the low-copy-number vector. This allows the investigation of the effects of different antigen amounts delivered to APC on the induction of antigen-specific T-cell responses. In addition, by employing different SPI1 and SPI2 effector proteins, it is also possible to translocate the antigen at different time points of the Salmonella infection cycle.

Efficient in vivo CD4 and CD8 T-cell priming depends on the level of chimeric p60 expression and the type III carrier protein used for antigen translocation. The potential of attenuated serovar Typhimurium expressing different hybrid p60 proteins to induce antigen-specific CD4 and CD8 T cells in vivo was investigated. For this purpose, BALB/c mice were orally immunized with a single dose of SB824 harboring the appropriate plasmid. Six weeks after inoculation, ELISPOT assays were performed to determine the frequencies of p60-specific CD4 and CD8 T cells. The frequencies of p60367-378-specific CD4 and p60217-225-specific CD8 T cells were calculated as the number of IFN-{gamma} spots generated per 105 spleen cells in the presence of the corresponding synthetic peptides. Mice immunized with SB824 expressing SopE2/p60 (pHR261) or SifA/p60 (pHR271) from medium-copy-number vectors revealed numbers of IFN-{gamma}-producing cells reactive with p60217-225 similar to those of mice immunized with SB824(pHR241) constitutively expressing YopE/p60 (Fig. 2). In contrast, immunization of animals with SB824(pHR262) or SB824(pHR272) expressing translocated SopE2/p60 or SifA/p60 from low-copy-number vectors did not induce measurable numbers of antigen-specific CD8 T cells. Interestingly, mice immunized with SB824 expressing SspH2/p60 from a medium-copy-number vector (pHR281) revealed not only a prominent p60-specific CD8 T-cell response but also high numbers of IFN-{gamma}-producing cells reactive with p60367-378 (Fig. 2). Contrary to the results observed in mice after immunization with Salmonella expressing SopE2/p60 or SifA/p60 from low-copy-number vectors, animals immunized with SB824 expressing SspH2/p60 from the low-copy-number vector pHR282 showed no significant differences in the number of CD8 T cells reactive with p60217-225 compared to mice immunized with SB824 carrying the medium-copy-number vector pHR281. However, the efficient induction of simultaneous p60-specific CD4 and CD8 T-cell responses was strictly dependent on the overexpression of SspH2/p60 from the medium-copy-number vector.



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  		FIG. 2. Frequencies of p60-specific CD4 and CD8 T cells in spleens of mice immunized with serovar Typhimurium strain SB824 bearing the indicated plasmids. T-cell frequencies were determined by ELISPOT assays as described in Materials and Methods. The frequencies of cells reactive with p60217-225 (filled bars) or p60367-378 (open bars) are shown as the number of reactive cells per 105 splenocytes. The standard deviations of three cultures from 15 individual mice per group are indicated. Values for measurable p60-specific CD8 T cells do not differ significantly (P > 0.05). The copy numbers of the respective plasmids are indicated (MC, medium-copy-number vector; LC, low-copy-number vector). Promoters are written in italics, and the respective carrier proteins fused to p60/M45 are indicated.

Simultaneous antigen-specific CD4 and CD8 T-cell priming is attributed to a "fine-tuned" overexpression of chimeric SspH2/p60. In order to determine the influence of the promoter and the particular carrier protein used for antigen delivery on the ability to induce concomitant CD4 and CD8 T-cell responses, we constructed the following medium-copy-number vectors (Table 1). Plasmid pHR273 bears the genetic information for SifA/p60/M45 under expression control of the sspH2 promoter, whereas plasmids pHR283 and pHR284 encode SspH2/p60/M45 under the control of the sifA promoter and the constitutively active lac promoter, respectively. Furthermore, to obtain information about the intrinsic properties of the carrier molecule itself, we changed the protein conformation by truncating SspH2, resulting in plasmid pHR285, which carries the genetic information for SspH21-151/p60/M45 under the control of its native promoter.

To determine the levels of expression and translocation of these constructs, we infected macrophages with Salmonella and performed biochemical fractionations as described above. As shown in Fig. 3, P388D1 cells infected with SB824(pHR273) expressing SifA/p60 under the control of the sspH2 promoter revealed decreased antigen concentrations compared to the expression under the control of the native sifA promoter (pHR271) (Fig. 1). However, the time course of protein production and translocation was comparable to the highest concentration of the fusion protein 24 h postinfection. Expression of SspH2/p60 under the control of the sifA promoter (pHR283) (Fig. 3) was also less efficient than the expression of chimeric SspH2 under the control of the sspH2 promoter (pHR281) (Fig. 1). As expected, expression of SspH2/p60 under the control of the lac promoter (pHR284) revealed constitutive production of the antigen at all three time points (Fig. 3), but interestingly, translocation of the fusion protein to the cytosol of macrophages was first observed after 6 h, reflecting the specific translocation by the SPI2 TTSS. In Fig. 3, it is demonstrated that the expression and translocation levels of the truncated hybrid SspH21-151/p60 protein by SB824(pHR285) were indistinguishable from those of SspH21-214/p60 (pHR281) (Fig. 1).



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  		FIG. 3. Fractionation of P388D1 cells infected with serovar Typhimurium strains expressing plasmid-encoded hybrid p60 proteins. Fractionations were carried out 2, 6, and 24 h after infection. Fraction 1, Triton X-100-insoluble fraction containing internalized bacteria; fraction 2, Triton X-100-soluble P388D1 cell lysate containing translocated proteins. The total protein amounts obtained from both fractions were loaded. Chimeric p60 proteins were detected by protein immunoblotting with a MAb to M45. The copy numbers of the respective plasmids are indicated (MC, medium-copy-number vector; LC, low-copy-number vector). Promoters are written in italics, and the respective carrier proteins fused to p60/M45 are indicated.

Immunization of BALB/c mice with SB824 carrying pHR273 (SifA/p60 under the control of the sspH2 promoter) or pHR283 (SspH2/p60/M45 under the control of the sifA promoter) led to the induction of a p60-specific CD8 T-cell response but not to a CD4 T-cell response (Fig. 4). In contrast, constitutive expression of SspH21-214/p60 encoded by plasmid pHR284 resulted in simultaneous antigen-specific CD8 and CD4 T-cell priming in immunized mice. The frequency of p60367-378-specific CD4 T cells was slightly, but not significantly, lower than in animals immunized with SB824(pHR281) (Fig. 2). To further elucidate the influence of the carrier protein itself on the observed induction of CD4 T cells and to exclude conformational reasons for this effect, we constructed plasmid pHR285, which encodes the truncated hybrid SspH21-151/p60 protein under the expression control of its native promoter. Vaccination of mice using this construct again induced simultaneous p60-specific CD8 and CD4 T-cell responses (Fig. 4), with T-cell frequencies indistinguishable from those of mice immunized with SB824(pHR281).



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  		FIG. 4. Frequencies of p60-specific CD4 and CD8 T cells in spleens of mice immunized with serovar Typhimurium strain SB824 bearing the indicated plasmids. T-cell frequencies were determined by ELISPOT assays as described in Materials and Methods. The frequencies of cells reactive with p60217-225 (filled bars) or p60367-378 (open bars) are shown as the number of reactive cells per 105 splenocytes. The standard deviations of three cultures from 15 individual mice per group are indicated. Values for measurable p60-specific CD4 and CD8 T cells do not differ significantly (P > 0.05). The copy numbers of the respective plasmids are indicated (MC, medium-copy-number vector; LC, low-copy-number vector). Promoters are written in italics, and the respective carrier proteins fused to p60/M45 are indicated.


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DISCUSSION
 
Efficient antigen display is an indispensable requirement for induction of T cells. The strength of antigen presentation generally depends on antigen access to the respective processing compartment, the antigen-processing efficacy, and antigen abundance (19, 53). Salmonella TTSS-mediated translocation can be used for efficient delivery of heterologous antigens to the cytosol of APC, leading to prominent CD8 T-cell priming in orally immunized mice (44). In this study, we directly compared the influences of SPI1- and SPI2-directed expressions of translocated hybrid p60 proteins on the induction of antigen-specific CD4 and CD8 T cells. Furthermore, the amount of the respective chimeric p60 protein to be delivered into the cytosol of APC was varied by using low- and medium-copy-number expression vectors. Immunoblot analyses of P388D1 cells infected with recombinant Salmonella strains revealed that SPI1- and SPI2-mediated translocations of hybrid proteins occurred at different time points during the bacterial infection cycle. As expected, SopE2/p60 derived from a low- or medium-copy-number plasmid under the control of its native promoter was detected in the cytosolic fractions of macrophages 2 and 6 but not 24 h after infection. In contrast, chimeric proteins engaged by SPI2 (SifA/p60 and SspH2/p60) were detectable after 6 and 24 h when medium-copy-number vectors were used and after 24 h only when low-copy-number plasmids were used. Interestingly, constitutive expression of SspH2/p60 by SB824(pHR284) 2 h after infection did not result in translocation of the fusion protein, indicating specific association of SspH2 with the SPI2 TTSS apparatus, which is maximally activated several hours after Salmonella invasion.

Oral immunization of mice with Salmonella strains expressing SopE2/p60, SifA/p60, or SspH2/p60 from medium-copy-number vectors led to the induction of comparable numbers of p60-specific CD8 T cells. Thus, sequential translocation of the model antigen within a period of 24 h after contact with APC did not influence the magnitude of the CD8 T-cell response. This result is in line with previous observations indicating that CD8 T cells are activated during the first 24 h of bacterial infection (31). However, for efficient CD8 T-cell priming, a relatively large amount of translocated SopE2/p60 or SifA/p60 was required, because mice immunized with Salmonella expressing these chimeric proteins from low-copy-number vectors did not reveal measurable numbers of p60-specific CD8 T cells. It is noteworthy that both types of recombinant plasmids (low- and medium-copy-number vectors) were remarkably stable in vivo, with ~95% of the bacterial population retaining the respective plasmid 10 days after infection (data not shown). Thus, the observed differences in the potential to induce p60-specific CD8 T cells were not due to plasmid instability.

In contrast to SopE2/p60 and SifA/p60, both translocated SspH2/p60 derived from a low-copy-number plasmid and SspH2/p60 derived from a medium-copy-number vector induced prominent CD8 T-cell priming. Moreover, the overexpression of SspH2/p60 under the control of the sspH2 promoter mediated by the medium-copy-number plasmid resulted in simultaneous p60-specific induction of CD4 T cells as well. We asked ourselves whether the observed CD4 T-cell response was dependent on the activity of the sspH2 promoter or on SspH2 itself. Expression of SifA/p60 under the control of the sspH2 promoter and expression of SspH2/p60 under the control of the sifA promoter resulted in moderate levels of chimeric proteins translocated into the cytosol of macrophages. In immunized mice, these antigen expression patterns induced a CD8 T-cell but not a CD4 T-cell response. However, when SspH2/p60 was expressed under the control of the constitutively active lac promoter on a medium-copy-number vector (pHR284), higher concentrations of the chimeric protein were detected in infected macrophages, leading to p60-specific CD8 and CD4 T-cell priming in immunized mice. Comparable results were obtained when truncated SspH21-151 was used to translocate p60. Under the expression control of its native sspH2 promoter on a medium-copy-number vector, this hybrid protein was efficiently produced and translocated, resulting in concomitant CD4 and CD8 T-cell responses. Taking these data together, the capability of the carrier protein SspH2 to direct p60 to the MHC class I-restricted, as well as to the MHC class II-restricted, processing pathway is a specific feature of the protein itself. A prerequisite for simultaneous CD4 and CD8 T-cell priming is the overexpression of hybrid SspH2, which can be achieved by using the native or the lac promoter for hybrid gene expression on a medium-copy-number vector. In further experiments, we fused >300 amino acids of listeriolysin O to SspH2 (data not shown). As demonstrated for SspH2/p60 proteins, overexpression of chimeric SspH2/LLO by Salmonella led to simultaneous induction of antigen-specific CD4 and CD8 T cells in orally immunized mice. Thus, the observed concomitant T-cell priming is not restricted to one antigen.

What is known about the function of SspH2? Miao et al. and Sory et al. employed the CyaA fusion protein strategy to examine SPI2 TTSS-dependent translocation of SspH2 (33, 47). Stationary-phase bacteria expressing SspH2/CyaA were unable to mediate increases in intracellular cyclic AMP after 1 h of infection but were able to cause elevation of the cyclic AMP level after 6 h of infection. These data are consistent with our results showing that hybrid SspH2/p60 protein translocation was detectable 6 h after infection. In a more recent publication, Miao et al. convincingly demonstrated that SspH2 localizes with the polymerizing actin cytoskeleton, most likely through an interaction with the actin cross-linking protein filamin (34). Further analysis revealed that the filamin-binding domain is located within the amino-terminal 61 residues of SspH2 (34). Like filamin, SspH2 displayed a subcellular localization pattern, being highly enriched in membrane ruffles and in the cytoplasm. In addition, SspH2 colocalized with vacuole-associated actin polymerizations induced by intracellular Salmonella (34, 32). However, these observations cannot explain how overexpressed SspH2/p60 gains access to the MHC class II presentation pathway. It is tempting to hypothesize that the overexpression of chimeric SspH2 leads to the saturation of the translocation process mediated by the envelope-associated organelle known as the needle complex (16). Thus, in addition to the delivery of hybrid SspH2 into the cytosol of host cells, it is conceivable that the protein is secreted through the flagellar export pathway into the SCV, followed by MHC class II processing. In fact, recently it has been proposed that some TTSS-exported proteins contain an ancestral flagellar secretion signal within their N-terminal amino acid sequences (28). In our laboratory, investigation is under way to define how overexpressed SspH2/p60 enters the MHC class II presentation pathway.

We emphasize that there is a need for simultaneous induction of CD4 and CD8 T cells as a strategy for vaccine development. For example, data from humans and animal models have indicated that CD4 and CD8 T cells are activated in response to Mycobacterium tuberculosis infection (12). This pathogen resides primarily in a vacuole within macrophages, resulting in MHC class II presentation of mycobacterial antigens to CD4 T cells (7). However, it has been demonstrated that certain M. tuberculosis antigens can be presented by MHC class I to CD8 T cells as well (29, 49). In addition to complex bacterial microbes, viruses and tumors are also known to be targeted by both CD4 and CD8 T cells. A key function in natural immune responses to viral pathogens and tumors is exerted by mature dendritic cells (DC) (20, 30). Activation of DC is achieved either by innate immunity triggers, such as microbial ligands (e.g., lipopolysaccharide) to Toll-like receptors (2), or by adaptive immunity triggers, such as CD40 ligand on activated CD4 T cells (4). Only properly matured DC will deliver the signals required for full induction of memory and effector CD8 T cells. Thus, induction, expansion, and maintenance of CD8 T-cell responses are achieved through well-balanced interactions between CD4 T cells, DC, and CD8 T cells (41).

We have identified a translocated SPI2 effector protein, SspH2, that can be efficiently used for heterologous protein delivery to APC, resulting in simultaneous induction of antigen-specific CD4 and CD8 T cells in orally vaccinated mice. In the future, SspH2-mediated translocation of foreign antigens may become a useful tool for the development of vaccines against microbial pathogens and tumors, which are known to be controlled by both CD4 and CD8 T cells.


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ACKNOWLEDGMENTS
 
The expert technical assistance of Jeannette Sauer is acknowledged.

H.R. was supported by the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm "Neue Vakzinierungsstrategien," grant RU 838/1-2).


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FOOTNOTES
 
* Corresponding author. Mailing address: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig Maximilians Universität München, Pettenkoferstr. 9a, 80336 München, Germany. Phone: 49-89-51605261. Fax: 49-89-51605248. E-mail: ruessmann{at}m3401.mpk.med.uni-muenchen.de. Back

Editor: J. T. Barbieri


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Infection and Immunity, January 2005, p. 334-341, Vol. 73, No. 1
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.1.334-341.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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