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Infection and Immunity, April 2008, p. 1590-1598, Vol. 76, No. 4
0019-9567/08/$08.00+0     doi:10.1128/IAI.00987-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Analysis of the Protective Capacity of Three Streptococcus suis Proteins Induced under Divalent-Cation-Limited Conditions

Jesús Aranda,¹ Maria Elena Garrido,^1,2 Pilar Cortés,^1,3 Montserrat Llagostera,^1,2 and Jordi Barbé^1,2*

Department de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain,¹ Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, 08193 Barcelona, Spain,² Servei d'Anàlisis i d'Aplicacions Microbiològiques, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain³

Received 19 July 2007/ Returned for modification 3 October 2007/ Accepted 2 January 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Streptococcus suis is a gram-positive pathogen that causes serious diseases in pigs and, in some cases, humans. Three genes of the virulent S. suis 89/1591 strain, encoding putative divalent-cation-binding lipoproteins, were isolated based on information obtained from the draft annotation files of this organism's genome. The products of these genes, which are inducible by divalent-cation deprivation, were subsequently purified, and their immunogenic and protective abilities were analyzed. All three proteins (SsuiDRAFT 0103, SsuiDRAFT 0174, and SsuiDRAFT 1237) were found to be immunogenic, but only one of them (SsuiDRAFT 0103) induced a significant protective response (87.5%, P = 0.01) against the same S. suis strain. Furthermore, the S. suis ssuiDRAFT 1240 gene (adcR), which encodes a predicted regulator of Zn²⁺ and/or Mn²⁺ uptake in streptococci, was cloned, and its protein product was purified. Electrophoretic mobility shift assays with purified S. suis AdcR protein showed experimentally, for the first time, that the AdcR DNA-binding sequence corresponds to the TTAACNRGTTAA motif. In addition, a requirement for either Zn²⁺ or Mn²⁺, but not Fe²⁺, to establish in vitro binding of AdcR to its target sequence and the ability of AdcR to bind the ssuiDRAFT 0103 and ssuiDRAFT 1237 gene promoters but not the promoter of the ssuiDRAFT 0174 gene were demonstrated. Taken together, these data suggest that SsuiDRAFT 0103 is a good candidate for vaccines against S. suis and support preliminary results indicating that bacterial envelope proteins involved in the uptake of divalent cations other than iron may be useful for protective purposes.

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Streptococcus suis is a significant gram-positive bacterium that causes important pathogenic entities, such as meningitis, septicemia, sudden death, and pneumonia, mainly in swine (30). Humans may also be infected by S. suis, usually after they have direct and extended contact with either infected or healthy carrier animals (1). To date, 35 different serotypes of S. suis have been described based on this bacterium's polysaccharide capsular antigen (21). Of these, serotype 2 has been shown to be the serotype most frequently involved in infectious diseases caused by S. suis (30). While several approaches to develop either live or recombinant vaccines to prevent S. suis-mediated disease have been tested (14, 30), putative protective strategies must still involve a wide range of weapons against this bacterium.

ATP-binding cassette (ABC) transporters are widespread in living organisms and comprise one of the largest protein families. In bacterial cells, these transporters import a variety of allocrites, including metal ions (9, 17). ABC transporters are multicomponent systems consisting of two membrane-inserted subunits, two components inside the cytoplasm that carry the ATP-binding site, and the substrate-specific binding protein located outside the cytoplasm (3, 11). In gram-negative bacteria, the binding protein is a soluble periplasmic protein. In gram-positive bacteria and archaea, it is a lipoprotein with a characteristic N-terminal lipid anchored to the cytoplasmic membrane. This lipoprotein is the ABC transporter component that is most exposed outside the cell surface (20, 31).

ABC transporters related to divalent-cation uptake have been shown to be involved in the virulence of many gram-negative and gram-positive bacteria (19, 22). Furthermore, several components of these ABC systems present in the cell wall, especially molecules such as lipoproteins, have immunogenic properties against the bacterial species from which they are derived (10, 23). For this reason, it has been suggested that these molecules have putative protective abilities (17), and this has been confirmed in some bacterial species, such as Yersinia pestis (34).

The entire genome of the S. suis 89/1591 virulent strain belonging to serotype 2 has been sequenced, and annotation of the sequence is near completion. Taking advantage of this fact, we searched the S. suis draft annotation database for genes encoding putative components of predicted divalent-cation-binding ABC transporters to analyze the immunogenicity and protective properties of these proteins, as well as the regulatory mechanism behind their expression.

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Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this work are listed in Table 1. Escherichia coli strains DH5 and BL21-CodonPlus(DE3)-RIL were grown in Luria-Bertani medium (26). When necessary, ampicillin (50 µg/ml), chloramphenicol (34 µg/ml), and 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Roche) were added to the growth media. S. suis strain 89/1591 was grown in Todd-Hewitt (TH) (Difco) medium supplemented with 2% yeast extract (Difco). All cultures were incubated at 37°C, and agitation was used for E. coli but not for S. suis. The divalent-cation chelator 2,2'-dipyridyl (DPD) (Sigma) was added at a final concentration of 150 µM to TH broth 30 min before inoculation of S. suis cells. DNA extraction, cloning, transformation, and other molecular techniques used in this work were carried out as previously described (29).

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TABLE 1. Bacterial strains and plasmids used in this study

Cloning, protein purification, and Western blot analysis. The S. suis proteins used in this work were selected based on their predicted functions in the draft annotation database for this organism (http://genome.ornl.gov/microbial/ssui/) and were expressed and purified as follows. DNA fragments containing genes encoding the desired proteins were amplified from purified chromosomal DNA of S. suis strain 89/1591 by PCR using the primers listed in Table 2. To facilitate restriction enzyme digestion of the PCR products, PCR primers were designed with one to five extra nucleotides at their 5' ends adjacent to the recognition site for the corresponding enzymes. Purified PCR products were then enzymatically digested with the corresponding restriction enzymes, cloned into the appropriate restriction sites in the polylinker of the pET15b expression vector, and transformed into E. coli DH5 cells. Recombinant plasmids were predicted to express an N-terminal His₆-tagged fusion protein, and correct in-frame fusions of the protein genes in pET15b were confirmed by sequencing plasmid DNA with the T7 Promoter and T7 Terminator primers (Macrogen Sequencing Service). Recombinant plasmids were then used to transform strain BL21-CodonPlus(DE3)-RIL.

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TABLE 2. Primers used in this study

An overnight culture of each resulting BL21-CodonPlus(DE3)-RIL strain was diluted (1/20) in 10 ml of Luria-Bertani medium and incubated at 37°C until the optical density at 600 nm (OD₆₀₀) was 0.6. Expression of the fusion proteins was induced at this time by addition of IPTG to a final concentration of 1 mM. After incubation for an additional 3 h at 37°C, the cells were collected by centrifugation at 8,000 x g for 10 min and resuspended in 1 ml of equilibration/wash buffer (50 mM sodium phosphate, 300 mM sodium chloride; pH 7) containing Complete Mini protease inhibitor cocktail (Roche). The cell suspensions were then lysed by sonication on ice for 5 min at 50 W by using a Braun LabsonicU (Braun Biotech International). Unbroken cells and debris were removed by centrifugation at 7,000 x g for 10 min. Each supernatant was subsequently mixed with 700 µl of BD TALON resin (Clontech) that had been previously equilibrated with 5 resin volumes of equilibration/wash buffer. Mixing was carried out by agitating the preparation for 20 min at room temperature, after which the resin was washed twice with 10 volumes of equilibration/wash buffer. Proteins were eluted with 1 ml of elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole). Finally, the purified proteins were dialyzed against 50 mM sodium phosphate (pH 7.0) to remove the imidazole and then visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (24).

Both the crude extracts of E. coli BL21-CodonPlus(DE3)-RIL cells overexpressing the proteins of interest and the purified proteins were visualized by Coomassie blue staining of sodium dodecyl sulfate-polyacrylamide gels. The antigenicity of the proteins was analyzed by Western blotting as described previously (18).

Mobility shift assays and DNase I footprinting. Electrophoretic mobility shift assays (EMSA) were performed as previously described (6), with slight modifications. Briefly, DNA promoters were PCR amplified from S. suis genomic DNA using suitable oligonucleotide primers (Table 2), and the purified PCR fragments were cloned into the pGEM-T vector. The presence of the desired promoter was confirmed by sequencing the plasmid DNA using the M13F-pUC(–40) and M13R-pUC(–40) primers of the pGEM-T vector. DNA probes were prepared by PCR amplification with one of the primers labeled with digoxigenin (DIG) at its 5' end and were purified. DNA-protein reaction mixtures (20 µl) containing 25 ng of a DIG-labeled DNA probe and either 0 or 10 µg of purified S. suis AdcR protein were incubated in EMSA buffer (20 mM Tris-HCl [pH 8], 50 mM KCl, 5% [vol/vol] glycerol, 1 µg bulk carrier sperm salmon DNA, 0.5 mM 1,4-dithiothreitol, 0.1 mg bovine serum albumin per ml) for 10 min at room temperature.

To determine whether AdcR binding was divalent cation dependent, 5x EMSA buffer was supplemented with EDTA at a final concentration of 1 mM in the presence or absence of either Zn₂SO₄, Mn₂SO₄, or Fe₂SO₄ (each at a final concentration of 1 mM). DNA-protein complexes were visualized by separation on a 5% nondenaturing polyacrylamide gel (40 mM Tris-acetate) at 150 V for 1.5 h and then transferred to a Biodine B nylon membrane (Pall Gelman Laboratory). DIG-labeled DNA-protein complexes were detected using the manufacturer's protocol (Roche). DNase I footprinting assays were carried out using an ALF sequencer (Amersham Biosciences) as described previously (7).

RNA techniques. RNA was isolated as described previously (15), with slight modifications. Briefly, 10-ml S. suis cultures in the mid-exponential growth phase (OD₆₀₀, 0.6) were collected by centrifugation at 8,000 x g for 10 min. The cells were then resuspended by vigorous vortexing at 37°C for 10 min in 300 µl of prelysis buffer containing 10 mg of lysozyme per ml. Total RNA was extracted by using an RNeasy mini kit (Qiagen) according to the manufacturer's instructions. DNA contamination was removed from the RNA during purification by treatment with RNase-free DNase (Qiagen), followed by digestion with DNase Turbo (Ambion). The concentration and integrity of the RNA were determined by measuring the OD₂₆₀ and by 1% agarose gel electrophoresis, respectively. Reverse transcriptase PCR (RT-PCR) assays were performed using a Titan One Tube RT-PCR system (Roche) by following the manufacturer's instructions. Real-time RT-PCR analysis of gene expression either in the presence or in the absence of the divalent-cation-chelating agent DPD was performed for all genes as reported previously (8); specific internal oligonucleotide primers were used for each gene (Table 2). In all cases, the absence of DNA in RNA samples was tested by performing PCR without RT.

Serum preparation and protection assays. Female BALB/cAnNHsd mice (3 weeks old) obtained from Harlan Iberica (Barcelona, Spain) were used for serum preparation and protection assays. All animals were quarantined for 1 week. A maximum of four animals per cage were housed under specific-pathogen-free conditions at 19 to 21°C with an artificial cycle consisting of 12 h of light and 12 h of darkness; the relative humidity was 50 to 60%. Antibiotic-free pelleted food and autoclaved water were provided ad libitum during the experiments. The animals were monitored daily for morbidity and mortality. All animal experiments were approved by the Universitat Autònoma de Barcelona Animal Ethics Committee.

Purified and dialyzed SsuiDRAFT 0103, SsuiDRAFT 0174, and SsuiDRAFT 1237 proteins were resuspended in 50 mM sodium phosphate (pH 7.0) and quantified by the Lowry method using bovine serum albumin as a protein standard. To prepare the vaccines, 10 µg of each protein per dose was adsorbed with Imject alum adjuvant (aluminum hydroxide; Pierce) according to the manufacturer's protocol.

To obtain serum for Western blot assays, six 4-week-old female BALB/cAnNHsd mice were inoculated with 0.1 ml of a TH broth suspension containing the 50% lethal dose (LD₅₀) (2.9 x 10⁶ CFU/animal) of strain S. suis 89/1591 (2) or 0.1 ml of TH broth alone as a negative control. Animals that survived until 30 days postinoculation were anesthetized with ketamine/xylacine. Blood was collected by cardiac puncture, incubated at 37°C for 2 h to facilitate clot formation, and then centrifuged at 300 x g for 10 min. Serum was recovered and stored at –20°C for Western blot assays.

For protection assays, three groups of eight female 4-week-old BALB/cAnNHsd mice were inoculated intraperitoneally with 0.1 ml of SsuiDRAFT 0103, SsuiDRAFT 0174, or SsuiDRAFT 1237 protein. Two additional groups of eight mice each inoculated with 0.1 ml of either 50 mM sodium phosphate (pH 7.0) or the same solution with Imject alum adjuvant as negative controls. After 2 weeks, a second immunization was carried out, followed 3 weeks later by a challenge consisting of intraperitoneal injection of 0.1 ml of a suspension containing 20 LD₅₀s of the S. suis 89/1591 strain in TH broth supplemented with 10% inactivated bovine serum. The survival of the mice was subsequently monitored for 21 days.

Statistical analysis. Fisher's exact test was used to analyze the statistical significance of the lethal challenge data.

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Organization and transcriptional analysis of genes encoding predicted divalent-cation-related ABC transporter components in S. suis. Three open reading frames (ORFs), whose products have been annotated as putative divalent-cation-binding lipoproteins (SsuiDRAFT 0103, SsuiDRAFT 0174, and SsuiDRAFT 1237) (Fig. 1A), are listed in the draft genome annotation database for the S. suis 89-1591 strain (http://genome.ornl.gov/microbial/ssui/). BLASTP analysis corroborated that the predicted products of these ORFs exhibit between 43 and 70% identity to zinc-binding (SsuiDRAFT 0103 and SsuiDRAFT 1237) or iron-binding (SsuiDRAFT 0174) streptococcal lipoproteins. Further study of the DNA surrounding sequences of these three ORFs by TBLASTN analysis established the putative genetic organization of the DNA regions (Fig. 1B and 1C). The hypothetical arrangements were confirmed by RT-PCR, which showed that a single transcriptional unit consisted of ORFs ssuiDRAFT 1240 to ssuiDRAFT 1237 (Fig. 1B) and that transcription of ORFs ssuiDRAFT 0173 to ssuiDRAFT 0176 yielded a single mRNA (Fig. 1C). In contrast, ORF ssuiDRAFT 0103 seems to be physically isolated since no ORF was detected immediately upstream or downstream of it in the contig. It should also be noted that TBLASTN analyses showed that the structures of the S. suis DNA regions containing either ORF ssuiDRAFT 0174 or ORF ssuiDRAFT 1237, as well as the transcriptional organization of ssuiDRAFT 0103 as a monocistronic unit, are preserved in other streptococcal species, such as Streptococcus pyogenes and Streptococcus agalactiae (data not shown). In fact, the protein products of ORFs ssuiDRAFT 1240, ssuiDRAFT 1239, ssuiDRAFT 1238, and ssuiDRAFT 1237 have high degrees of identity (52, 77, 82, and 70%, respectively) with the protein products encoded by Streptococcus pneumoniae adcR, adcC, adcB, and adcA, all of which are involved in Zn²⁺ and/or Mn²⁺ uptake in that organism and in Streptococcus gordonii (12, 25). Furthermore, these genes have been shown to be required for transformation competence of S. pneumoniae and biofilm formation by S. gordonii (12, 25). It is also noteworthy that transcriptional units containing ORFs ssuiDRAFT 0103, ssuiDRAFT 0174, and ssuiDRAFT 1237 are inducible in the presence of DPD, as would be expected if all three ORFs belong to the divalent-cation uptake systems of S. suis (Fig. 2).

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FIG. 1. (A) ORFs of the S. suis 89/1591 genome whose products have been predicted to be involved in divalent-cation uptake according to the provisional annotation of the genome of this organism. The lengths of the gene products (in amino acids [aa]) and the putative functions of the genes are also indicated. (B and C) Genetic organization, as determined by RT-PCR analysis of RNA, of S. suis 89/1591 chromosomal regions containing ssuiDRAFT 1237 (B) and ssuiDRAFT 0174 ORFs (C). The large filled arrows indicate the putative transcriptional units that have been tested. The primer sets used for the RT-PCR analyses are indicated by small arrows. RT-PCRs were carried out in the presence of total RNA (lanes A), in the presence of DNA (lanes B), or in the absence of both RNA and DNA (lanes C). BstEII-digested DNA was used as molecular size marker (lane MW).

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FIG. 2. DPD-mediated induction of several S. suis 89/1591 ORFs in the transcriptional units that include the ssuiDRAFT 0103, ssuiDRAFT 0174, and ssuiDRAFT 1237 ORFs. The induction factor is the ratio of the mRNA concentration of a gene from cells treated with DPD (150 µM) to the mRNA concentration of the gene from cells not treated with DPD. RNA was extracted from DPD-treated and nontreated cultures at an OD600 of 0.6. The amount of mRNA of each gene was determined by using a standard curve generated by amplification of an internal fragment of the S. suis ssuiDRAFT 0855 ORF encoding the cysteine synthase enzyme, whose expression is not sensitive to divalent-cation deprivation. The results are the means of two independent experiments (each carried out in duplicate); the standard deviations are indicated by error bars.

AdcR protein controls ssuiDRAFT 0103 and ssuiDRAFT 1237 transcriptional units. As shown in Fig. 1B, ssuiDRAFT 1240 (homologous to the adcR gene of the streptococci) is the first gene in the transcriptional unit containing ORF ssuiDRAFT 1237. The product of the adcR gene is a repressor that controls the uptake of Zn²⁺ and/or Mn²⁺ in several gram-positive bacteria (9, 13, 25). Although an in silico comparison of sequences predicted a putative AdcR-binding motif (28), to our knowledge there is no experimental evidence effectively demonstrating the DNA recognition sequence of AdcR. For this reason, the S. suis AdcR protein was purified, and its ability to bind the promoter region of its own encoding transcriptional unit was analyzed by EMSA using a DNA fragment extending from position 50 to position –146 (with respect to the predicted translational starting point) as a probe (Fig. 3A). The EMSA results showed that the AdcR protein specifically binds to the promoter of its gene (Fig. 3B) and that the presence of either Zn²⁺ or Mn²⁺ is required for the specific binding of AdcR in vitro (Fig. 3C), whereas addition of Fe²⁺ did not promote DNA-protein complex formation (data not shown).

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FIG. 3. (A) Schematic diagram of the adcR gene with its promoter region (PadcR). The small arrows indicate the primer pair used to amplify the promoter in EMSA experiments. (B) Electrophoretic mobility of the DNA fragments containing the S. suis adcR promoter in the absence (lane 1) or in the presence (lane 2) of purified S. suis AdcR. The effects of a 300-fold molar excess of unlabeled S. suis adcR promoter (lane 3) or pGEM-T plasmid DNA (lane 4) on the migration of the S. suis adcR promoter in the presence of purified AdcR are also shown. (C) Effects of EDTA addition (1 mM) on the AdcR-binding capacity in the absence of exogenous divalent cation (lane 2) or in the presence of either 1 mM Zn2+ (lane 3) or 1 mM Mn2+ (lane 4). As a control, the mobility of the same promoter without AdcR is also shown (lane 1).

A footprinting experiment with the same DNA fragment that was used for the EMSA revealed that a core region consisting of 24 nucleotides was protected by AdcR binding (Fig. 4A). It is also worth noting that inside the AdcR-protected core region, one exact copy (TTAACAAGTTAA) and another very similar copy (TTAAAAGGTTAA) of the in silico-predicted AdcR-binding sequence (TTAACYRGTTAA) were found at positions –28 and –16, respectively. Analysis of the role of each of these two motifs in AdcR binding showed that the presence of the first motif (at position –28) is absolutely necessary for AdcR binding, whereas the second motif (at position –16) is dispensable (data not shown). Single substitutions of each of the nucleotides of the first motif (AdcR box) provided a more precise identification of the AdcR-binding sequence as TTAACNRGTTAA. In addition, insertion of a single nucleotide between the two central adenines of the TTAACAAGTTAA motif was sufficient to abolish AdcR binding (Fig. 4B).

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FIG. 4. (A) DNase I footprinting assays with coding and noncoding Cy5-labeled strands of the DNA fragment containing the S. suis adcR promoter in the absence or presence of purified AdcR from the same organism. The putative translational starting codon is indicated by bold type and underlining. The arrows indicate the transcriptional direction of the strands. (B) Single-nucleotide substitutions in the TTAACAAGTTAA sequence (AdcR Box) and their effects on the electrophoretic mobility of the S. suis adcR promoter. The wild type (wt), used as a positive control, consisted of the same fragment without nucleotide substitutions. The plus sign indicates the position at which an additional nucleotide (+N1) was added. Inverted arrows indicate the palindromic motifs. The deduced consensus sequence is also shown.

Moreover, and in agreement with the fact that only one of these motifs is necessary for AdcR binding, AdcR caused a delay in the electrophoretic mobility of the ssuiDRAFT 0103 ORF promoter, which contains only one copy of the AdcR-like binding sequence (TTAACTGGTTAA) (Fig. 5). By contrast, but as expected, AdcR was unable to bind the promoter of the transcriptional unit containing ssuiDRAFT 0174 because its lacked the AdcR-like recognition sequence (data not shown). Given that ssuiDRAFT 0174 was inducible in the presence of DPD, there must be an additional regulatory system for genes involved in the uptake of divalent cations other than Zn²⁺ and Mn²⁺ in S. suis.

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FIG. 5. (A) Schematic diagram of the ssuiDRAFT 0103 gene with its promoter region (P0103). The small arrows indicate the primer pair used to amplify the promoter in EMSA experiments. (B) Electrophoretic mobility of the DNA fragment containing the S. suis ssuiDRAFT 0103 promoter in the absence (lane 1) or in the presence (lane 2) of purified S. suis AdcR. The effects of a 300-fold molar excess of unlabeled S. suis ssuiDRAFT 0103 promoter (lane 3) and pGEM-T plasmid DNA (lane 4) on the migration of the S. suis ssuiDRAFT 0103 promoter in the presence of purified AdcR are also shown.

Immunogenic and protective capacities of the SsuiDRAFT 0103, SsuiDRAFT 0174, and SsuiDRAFT 1237 proteins. The SsuiDRAFT 0103, SsuiDRAFT 0174, and SsuiDRAFT 1237 proteins were overexpressed, purified, and analyzed on Western blots by using immunological reactive serum obtained from surviving mice after inoculation with S. suis wild-type strain 89/1591. The results showed that all three proteins triggered the synthesis of specific antibodies in mice infected with the pathogen (Fig. 6), whereas negative results were obtained when these proteins were tested with serum from noninfected mice (data not shown).

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FIG. 6. Western blot analysis with serum recovered from mice infected with the S. suis 89/1591 strain. The analysis was carried out with crude extracts from E. coli BL21-CodonPlus(DE3)-RIL cells containing the pET15b vector either alone as a negative control (lane 1) or overexpressing the SsuiDRAFT 0103 (lane 2), SsuiDRAFT 0174 (lane 3), and SsuiDRAFT 1237 (lane 4) proteins.

In order to determine whether the induction of this immunogenic response was sufficient to protect mice against S. suis infection, three groups of eight BALB/cAnNHsd mice were immunized twice with 10 µg of each of the three proteins. When the animals were inoculated with 20 LD₅₀s of the S. suis 89/1591 strain, practically all the mice died within 21 days after infection in the control group and in the group vaccinated with SsuiDRAFT 0174 (Fig. 7). Moreover, the viability of the mice inoculated with SsuiDRAFT 1237 was 50%, whereas the survival of animals vaccinated with SsuiDRAFT 0103 was dramatically higher (87.5%, P = 0.01) during the period of study (Fig. 7).

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FIG. 7. Survival curves for mice inoculated with solvent (*) or solvent with adjuvant () and vaccinated with purified SsuiDRAFT 103 (), SsuiDRAFT 174 (), or SsuiDRAFT 1237 () protein (eight mice per group). After the last immunization, the mice were challenged intraperitoneally with an amount of wild-type S. suis 89/1591 cells equivalent to 20 LD50s of this bacterial strain.

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Several regulatory transcriptional factors have been reported to be involved in the uptake of Zn²⁺ by different groups of bacterial species. The first of these regulators to be described was the E. coli Zur protein (zinc uptake regulator), which is widespread in gammaproteobacteria and alphaproteobacteria (28). A second Zn²⁺ uptake regulator is that of the Bacillus group and is also called Zur (16). The third and most recently recognized Zn²⁺ regulator to be described is AdcR, which is present in Streptococcus (28).

In this work, after searching the draft annotation database of the S. suis 89/1591 strain genome, we identified three genes (ssuiDRAFT 0103, ssuiDRAFT 0174, and ssuiDRAFT 1237) encoding putative components of divalent-cation uptake-related ABC transporters. The ORF belonging to the transcriptional unit containing one of these three genes and encoding AdcR was identified. This protein had been demonstrated previously to be an in vivo repressor controlling the genes involved in Zn²⁺ and/or Mn²⁺ incorporation in streptococci (9, 13, 25), and a putative recognition motif was proposed following in silico analysis (28). Data reported here effectively demonstrated, for the first time, not only that the in silico-predicted motif (TTAACYRGTTAA) is very similar to the in vitro functional binding sequence (TTAACNRGTTAA) but also that AdcR binding to its target sequence is dependent, at least in vitro, on the presence of either Zn²⁺ or Mn²⁺ but not on the presence of Fe²⁺. Likewise, our results indicated that the distance between the two palindromic halves of the AdcR-binding sequence must be strictly preserved. Nonetheless, as described above, despite the close relationship among all gram-positive bacteria, the regulation of Zn²⁺ uptake differs dramatically with respect to DNA recognition motifs. This finding suggests two different origins for Zn²⁺ uptake regulatory proteins in this bacterial phylum.

The results presented in this work also demonstrated that the product of the ssuiDRAFT 0103 gene belonging to the S. suis adcR regulon, which constitutes a single transcriptional unit, not only is immunogenic but also confers a statistically significant level of protection against S. suis challenge to mice vaccinated with this protein. Two other S. suis envelope proteins (SsuiDRAFT 0174 and SsuiDRAFT 1237), encoded in two independent polycistronic transcriptional units, with antigenic capacity but less protective power than the SsuiDRAFT 0103 protein were also identified. The reduced protective ability of these two proteins might be due, for example, to a less important role in divalent-cation uptake, or their exposure to the external medium may not be as extensive as that of the ssuiDRAFT 0103 ORF. However, since animals inoculated with S. suis produced antibodies against the three proteins, a combination or even only one of them might form the basis of a simple serological test for detecting S. suis infections on animal farms. Furthermore, it must be noted that although the expression of the ssuiDRAFT 0103, ssuiDRAFT 0174, and ssuiDRAFT 1237 ORFs was induced in the presence of the chelating agent DPD, only the ssuiDRAFT 0103 and ssuiDRAFT 1237 ORFs were under AdcR control. These data suggest that there is a second regulator of divalent-cation uptake, which controls at least ssuiDRAFT 0174 expression.

It is well known that many bacterial envelope proteins involved in iron uptake are immunogenic and that some of them are protective (4, 5). Similar results have been reported for some divalent-cation uptake systems other than the iron uptake systems (27, 32, 33). Nevertheless, data presented in this work are the first data to demonstrate that proteins belonging to the adcR regulon may be useful as vaccines. Finally, the fact that TBLASTN analysis confirmed that genes homologous to the ssuiDRAFT 0103 ORF are present in practically all streptococci for which sequence data are available (data not shown) suggests that the encoded product can be used as a tool to achieve broad protection against this significant pathogenic group of gram-positive bacteria.

 
This work was funded by grant AGL2005-03574 from the Ministerio de Educación y Ciencia (MEC) de España and by grant 2005SGR-533 from the Departament d'Universitats, Recerca i Societat de la Informació (DURSI) de la Generalitat de Catalunya. J. Aranda was a recipient of a predoctoral fellowship from the Universitat Autònoma de Barcelona.

We thank Joan Ruiz for his excellent technical assistance.

 
* Corresponding author. Mailing address: Department de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain. Phone: 34-93-581 1837. Fax: 34-93-581 2387. E-mail: jordi.barbe{at}uab.cat

Published ahead of print on 22 January 2008.

Editor: V. J. DiRita

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1
1. Arends, J. P., and H. C. Zanen. 1988. Meningitis caused by Streptococcus suis in humans. Rev. Infect. Dis. 10:131-137.[Medline]
2
2. Beaudoin, M., R. Higgins, J. Harel, and M. Gottschalk. 1992. Studies on a murine model for evaluation of virulence of Streptococcus suis capsular type 2 isolates. FEMS Microbiol. Lett. 78:111-116.[Medline]
3
3. Boos, W., and J. M. Lucht. 1996. Periplasmic binding protein-dependent ABC transporters, p. 1175-1209. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, DC.
4
4. Bosch, M., M. E. Garrido, A. M. Pérez de Rozas, I. Badiola, J. Barbé, and M. Llagostera. 2004. Pasteurella multocida contains multiple immunogenic haemin- and haemoglobin-binding proteins. Vet. Microbiol. 99:103-112.[CrossRef][Medline]
5
5. Brown, J. S., A. D. Ogunniyi, M. C. Woodrow, D. W. Holden, and J. C. Paton. 2001. Immunization with components of two iron uptake ABC transporters protects mice against systemic Streptococcus pneumoniae infection. Infect. Immun. 69:6702-6706.[Abstract/Free Full Text]
6
6. Bsat, N., and J. D. Helmann. 1999. Interaction of Bacillus subtilis Fur (ferric uptake repressor) with the dhb operator in vitro and in vivo. J. Bacteriol. 181:4299-4307.[Abstract/Free Full Text]
7
7. Campoy, S., M. Fontes, S. Padmanabhan, P. Cortés, M. Llagostera, and J. Barbé. 2003. LexA-independent DNA damage-mediated induction of gene expression in Myxococcus xanthus. Mol. Microbiol. 49:769-781.[CrossRef][Medline]
8
8. Campoy, S., G. Mazón, A. R. Fernández de Henestrosa, M. Llagostera, P. B. Monteiro, and J. Barbé. 2002. A new regulatory DNA motif of the gamma subclass Proteobacteria: identification of the LexA protein binding site of the plant pathogen Xylella fastidiosa. Microbiology 148:3583-3597.[Abstract/Free Full Text]
9
9. Claverys, J. P. 2001. A new family of high-affinity ABC manganese and zinc permeases. Res. Microbiol. 152:231-243.[Medline]
10
10. Cockayne, A., P. J. Hill, N. B. L. Powell, K. Bishop, C. Sims, and P. Williams. 1998. Molecular cloning of a 32-kilodalton lipoprotein component of a novel iron-regulated Staphylococcus epidermidis ABC transporter. Infect. Immun. 66:3767-3774.[Abstract/Free Full Text]
11
11. Dean, M., and R. Allikmets. 1995. Evolution of ATP-binding cassette transporter genes. Curr. Opin. Genet. Dev. 5:779-785.[CrossRef][Medline]
12
12. Dintilhac, A., G. Alloing, C. Granadel, and J.-P. Claverys. 1997. Competence and virulence of Streptococcus pneumoniae: Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases. Mol. Microbiol. 25:727-739.[CrossRef][Medline]
13
13. Dintilhac, A., and J.-P. Claverys. 1997. The adc locus, which affects competence for genetic transformation in Streptococcus pneumoniae, encodes an ABC transporter with a putative lipoprotein homologous to a family of streptococcal adhesins. Res. Microbiol. 148:119-131.[Medline]
14
14. Fittipaldi, N., J. Harel, B. D'Amours, S. Lacouture, M. Kobisch, and M. Gottschalk. 2007. Potential use of an unencapsulated and aromatic amino acid-auxotrophic Streptococcus suis mutant as a live attenuated vaccine in swine. Vaccine 25:3524-3535.[CrossRef][Medline]
15
15. Fontaine, M. C., J. Pérez-Casal, and P. J. Willson. 2004. Investigation of a novel DNase of Streptococcus suis serotype 2. Infect. Immun. 72:774-781.[Abstract/Free Full Text]
16
16. Gaballa, A., and J. D. Helmann. 1998. Identification of a zinc-specific metalloregulatory protein, Zur, controlling zinc transport operons in Bacillus subtilis. J. Bacteriol. 180:5815-5821.[Abstract/Free Full Text]
17
17. Garmory, H. S., and R. W. Titball. 2004. ATP-binding cassette transporters are targets for the development of antibacterial vaccines and therapies. Infect. Immun. 72:6757-6763.[Free Full Text]
18
18. Garrido, M. E., M. Bosch, R. Medina, A. Bigas, M. Llagostera, A. M. Pérez de Rozas, I. Badiola, and J. Barbé. 2003. fur-independent regulation of the Pasteurella multocida hbpA gene encoding a haemin-binding protein. Microbiology 149:2273-2281.[Abstract/Free Full Text]
19
19. Garrido, M. E., M. Bosch, R. Medina, M. Llagostera, A. M. Pérez de Rozas, I. Badiola, and J. Barbé. 2003. The high-affinity zinc-uptake system znuABC is under control of the iron-uptake regulator (fur) gene in the animal pathogen Pasteurella multocida. FEMS Microbiol. Lett. 221:31-37.[CrossRef][Medline]
20
20. Hantke, K. 2001. Bacterial zinc transporters and regulators. Biometals 14:239-249.[CrossRef][Medline]
21
21. Higgins, R., M. Gottschalk, M. Jacques, M. Beaudain, and J. Henrichsen. 1995. Description of six new capsular types (29-34) of Streptococcus suis. J. Vet. Diagn. Investig. 7:405-406.[Free Full Text]
22
22. Janulczyk, R., S. Ricci, and L. Björck. 2003. MtsABC is important for manganese and iron transport, oxidative stress resistance, and virulence of Streptococcus pyogenes. Infect. Immun. 71:2656-2664.[Abstract/Free Full Text]
23
23. Jomaa, M., J. Yuste, J. C. Paton, C. Jones, G. Dougan, and J. S. Brown. 2005. Antibodies to the iron uptake ABC transporter lipoproteins PiaA and PiuA promote opsonophagocytosis of Streptococcus pneumoniae. Infect. Immun. 73:6852-6859.[Abstract/Free Full Text]
24
24. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.[CrossRef][Medline]
25
25. Loo, C. Y., K. Mitrakul, I. B. Voss, C. V. Hughes, and N. Ganeshkumar. 2003. Involvement of the adc operon and manganese homeostasis in Streptococcus gordonii biofilm formation. J. Bacteriol. 185:2887-2900.[Abstract/Free Full Text]
26
26. Miller, J. H. 1992. A short course in bacterial genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
27
27. Ogunniyi, A. D., R. L. Folland, D. E. Briles, S. K. Hollingshead, and J. C. Paton. 2000. Immunization of mice with combinations of pneumococcal virulence proteins elicits enhanced protection against challenge with Streptococcus pneumoniae. Infect. Immun. 68:3028-3033.[Abstract/Free Full Text]
28
28. Panina, E. M., A. A. Mironov, and M. S. Gelfand. 2003. Comparative genomics of bacterial zinc regulons: enhanced ion transport, pathogenesis, and rearrangements of ribosomal proteins. Proc. Natl. Acad. Sci. USA 100:9912-9917.[Abstract/Free Full Text]
29
29. Sambrook, J., and D. W. Russell. 2001. Molecular cloning; a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
30
30. Staats, J. J., I. Feder, O. Okwumabua, and M. M. Chengappa. 1997. Streptococcus suis: past and present. Vet. Res. Commun. 21:381-407.[CrossRef][Medline]
31
31. Sutcliffe, I. C., and R. R. Russell. 1995. Lipoproteins of gram-positive bacteria. J. Bacteriol. 177:1123-1128.[Free Full Text]
32
32. Tai, S. S. 2006. Streptococcus pneumoniae protein vaccine candidates: properties, activities and animal studies. Crit. Rev. Microbiol. 32:139-153.[CrossRef][Medline]
33
33. Talkington, D. F., B. G. Brown, J. A. Tharpe, A. Koenig, and H. Russell. 1996. Protection of mice against fatal pneumococcal challenge by immunization with pneumococcal surface adhesin A (PsaA). Microb. Pathog. 21:17-22.[CrossRef][Medline]
34
34. Tanabe, M., H. S. Atkins, D. N. Harland, S. J. Elvin, A. J. Stagg, O. Mirza, R. W. Titball, B. Byrne, and K. A. Brown. 2006. The ABC transporter protein OppA provides protection against experimental Yersinia pestis infection. Infect. Immun. 74:3687-3691.[Abstract/Free Full Text]

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Infection and Immunity, April 2008, p. 1590-1598, Vol. 76, No. 4
0019-9567/08/$08.00+0     doi:10.1128/IAI.00987-07
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