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

Bacterial strains, plasmids, and growth conditions.Bacterial strains and plasmids used in this work are listed in Table 1. Escherichia coli strains DH5α 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).

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 DH5α 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.

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 × 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 × 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, 5× 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 × 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 × 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 × 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.

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 λ 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.