Abstract

BackgroundStreptococcus suis serotype 2 (SS2) has evolved into a highly infectious entity, posing a great threat to public health. Screening for and identification of protective antigens plays an important role in developing therapies against SS2 infections

MethodsMultiple strategies were used to investigate a new surface protein that has the potential to be a protective antigen. These strategies included molecular cloning, biochemical and biophysical analyses, enzymatic assay, immunological approaches (eg, immunoelectron microscopy), and experimental infections of animals

ResultsWe identified an enolase gene from SS2 and systematically characterized its protein product, enolase. Biophysical data indicated that S. suis enolase is an octameric protein. Enzymatic assays verified its ability to catalyze the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate. In consideration of the strong antigenicity of enolase, an efficient enolase-based method was established for monitoring SS2 infections. Combined evidence strongly indicated that SS2 enolase can localize on the bacterial cell surface and facilitate bacterial adherence. Additionally, we found that enolase can confer complete protection against SS2 infection to mice, which suggests that enolase has potential as a vaccine candidate

ConclusionsWe conclude that S. suis enolase functions as a protective antigen displayed on the bacterial cell surface and that it can be used to develop new strategies to combat SS2 infections

Streptococcus suis is an important swine pathogen and emerging zoonotic agent [1]. In total, 35 serotypes (1–34 and 1/2) have been proposed, of which serotype 2 is generally highly pathogenic and frequently isolated from diseased pigs [2]. In North America and the United Kingdom only sporadic cases of Streptococcus suis serotype 2 (SS2) infection have been known to occur in humans [3], whereas in China 2 large-scale outbreaks of human SS2 infections with clinical manifestation of streptococcal toxic shock syndrome (STSS) challenged the public health [4]. Very recently, STSS was found to be associated with the capability of virulent SS2 strains in China to stimulate the proliferation of T cells, naive T cells, and peripheral blood mononuclear cells [5]. In addition to the well-known SS2 outbreaks, there was also a recurrence of human SS2 meningitis in 2007 (recently documented by Feng et al [6]), which indicates the circulation of heterogeneous SS2 populations in China

Full understanding of the factors associated with virulence is a prerequisite for the search for new strategies targeting the SS2 challenge [1, 7, 8]. The large-scale outbreak of human SS2 infection in 2005 accelerated basic research in the SS2 field [9]. Our research group (the Beijing-Nanjing Joint SS2 Research Group) sequenced the whole genomes of 2 Chinese SS2 isolates (98HAH12 and 05ZYH33) [10] and discovered a suspected pathogenicity island, 89K [10, 11]. A genetic study demonstrated that a 2-component signal transduction system (SalK/SalR) from 89K is required for full virulence of the Chinese SS2 strain [11]. In addition, we determined that sortase A, a transpeptidase, contributes to high pathogenicity of S. suis [12]. This observation accords with that reported by Vanier's group [13]. Recently, Fittipalti et al [14] found that modification of peptidoglycan by N-deacetylation is an important factor in S. suis virulence. However, Zur, a zinc-responsive regulator, is not involved in SS2 pathogenesis [15], which is distinct from that of other pathogens (eg, Xanthomonas [16]). The mysterious mask on the highly pathogenic Chinese SS2 variants is gradually removed as our knowledge about this pathogen grows

Increasing amounts of accumulated data supported the hypothesis that surface-related proteins are involved in bacterial pathogenesis, exemplified by adhesion, invasion, and bacterial defense mechanisms [17–22 ]. Unexpectedly, multifunctional glycolytic enzymes (such as glyceraldehyde-3-phosphate dehydrogenase and α-enolase), which generally localize in the cytoplasm, were found to be exported to the cell surface of a variety of microorganisms by an unknown mechanism [23–28 ]. In fact, these enzymes play critical roles in bacterial colonization, persistence, and invasion of host tissue, in addition to their innate glycolytic and metabolic enzyme activities [29]. Enolase, an enzyme responsible for the dehydration of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP), was recognized recently as an immunodominant antigen involved in the virulence of Streptococcus species [24, 27, 30, 31]

In this study we identify and characterize a functional enolase gene homologue from STSS-causing SS2. In light of the significance of enolase as a novel antigen, we also systematically discuss its role in SS2 infections

Materials and Methods

Strains and plasmidsSS2 (strain 05ZYH33) was grown in Todd-Hewitt broth [15]. Escherichia coli (strains DH5α and BL21 [DE3]) was maintained in Luria-Bertani medium. The vectors pMD18-T (Takara) and pET28(a) (Novagen) were used for polymerase chain reaction cloning and protein expression in vitro, respectively

Bioinformatics-based analysesMultiple alignment analyses of enolase proteins (FJ895346) were performed using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and the output result was further processed with ESPript, version 2.2 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi), to generate the final version for Basic Local Alignment Search Tool for Proteins (BLASTP) [15]. The alignment analyses revealed the important amino acid residues that are conserved between the Streptococcus enolase homologues and the crystal structure of Streptococcus pneumonia enolase (Protein Data Bank entry 1W6T), which was used as a reference. To gain insight into the structure of enolase, structural modeling was performed using CPHmodels (http://www.cbs.dtu.dk/services/CPHmodels/), and then the relevant structure diagram was generated by means of Visual Molecular Dynamics. To further investigate the possible mechanism underlying the strong immunogenicity of S. suis enolase, we conducted B cell epitope prediction, as well as major histocompatibility complex II (MHC II)-restricted peptide analyses. BepiPred, version 1.0 (http://www.cbs.dtu.dk/services/BepiPred), which depends on the combination of a hidden Markov model and a propensity scale method, was applied to predict the location of linear B cell epitopes (score threshold for epitope assignment, 0.7). Similarly, we used NetMHCII, version 1.0 (http://www.cbs.dtu.dk/services/NetMHCII), to predict the binding of peptides to a number of different HLA-DR alleles by use of position-specific weight matrices. The prediction values were given as median inhibitory concentrations in units of nanomoles per liter, and strong and weak binding peptides were indicated in the output

Cloning and expression of S. suisenolaseA pair of specific primers were designed for amplification of the enolase gene (eno-F, 5′-GCGGATCCATGTCAATTATTACTG-3′; and eno-R, (5′-GCCTCGAGTTATTTTTTCAAGTTGTAG-3′) [32]. The underlined sequences are BamHI and XhoI, respectively. After pMD-18T cloning and direct sequencing, eno was inserted into pET28(a) via BamHI and XhoI, generating pET28::eno

E. coli BL21 transformed with pET28::eno was induced at 37°C for 4 h with the addition of 1 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG). After sonication, bacterial lysate was subjected to centrifugation for removal of the insoluble pellets. The acquired supernatant was filtered through a 0.22 μm-pore filter (Millipore) and applied on a His-trap column (Qiagen). The recombinant enolase was eluted with elution buffer containing 100 mmol/L imidazole. It was further purified via fast-phase liquid chromatography (FPLC) and visualized by means of 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

Enzymatic assays for enolaseTo address the capability of converting 2-PGE to PEP by enolase, different amounts of 2-PGE (0.25–12 mmol/L) were preincubated at 37°C for 3 min using 5 μg of purified enolase protein in HEPES buffer (100 mmol/L HEPES, 10 mmol/L MgCl2, and 7.7 mmol/L KCl; pH, 7.0). The release of PEP was measured at 240 nm on a spectrophotometer

Preparation of antienolase serumPolyclonal antibody against enolase was prepared routinely by immunizing rabbits subcutaneously at multiple sites with approximately 1 mg/kg of purified protein emulsified with Freund complete adjuvant (1:1). After 2 weeks each rabbit received 1 booster injection with the same antigen concentration emulsified with Freund incomplete adjuvant (1:1). Then serum samples were collected when the second booster injection was administered 7 d later

SDS-PAGE and Western blotSDS-PAGE and Western blot analyses were performed as described elsewhere [33]. The first antibody used was convalescent-phase swine serum from specific pathogen-free pigs that survived clinical infection with the SS2 strain 05ZYH33. The second antibody used was peroxidase-conjugated goat anti-swine immunoglobulin G (IgG) (Sigma)

Enzyme-linked immunosorbent assay (ELISA)ELISA was performed to test for the presence of enolase in S. suis. First, bacterial cells were used to coat ELISA plates (Greiner Bio-One). We included whole bacterial cells, sonication lysate, and cytoplasm samples. (Of note, SS2 bacterial cells collected by centrifugation were subjected to sonication after 30 min of treatment with 0.5 mg/mL lysozyme [Sigma] at room temperature.) After blocking with 1% bovine serum albumin, the plates were probed with serial dilutions of rabbit preimmunization serum and rabbit antienolase serum. The antibody was labeled with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma), which was in turn detected with O-phenylenediamine pnitrophenyl (Amresco). The value was recorded with a mode 550 enzyme-labeled instrument (Bio-Rad) at 490 nm

We also applied the enolase-based ELISA to probe whether enolase is associated with SS2 infection. Serum samples from specific pathogen-free pigs were used as negative control samples, and positive samples were collected from convalescent-phase piglets surviving infection with SS2 strain 05ZYH33. Serum samples from animals with suspected SS2 infection were collected from pig farms in Jiangsu Province, China [33]. The absorbance score was recorded at 490 nm in a microplate reader

Immunoelectron microscopy and immunofluorescence assayImmunoelectron microscopy was used to obtain physical evidence that enolase can be displayed on the bacterial cell surface. In accordance with the protocol described by Li et al [34], 70-nm ultrathin slices of SS2 bacterial cells were prepared and placed on nickel grids. After lysin treatment, the grids were floated on a blocking solution of phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin for 30 min and then incubated with a 1:50 dilution of rat antienolase polyclonal antibody at 4°C overnight. (Preimmunization rat serum was used as the control group.) A 1:5 dilution of goat anti-rat antibody conjugated to 10-nm gold particles (Sigma) was subsequently added and kept for 2 h. After 2 rounds of washing with PBS, separation by incubation in 2% glutaraldehyde, and 1 round of Formvar coating, slices were visualized by means of a Philips 301 transmission electron microscope

Immunofluorescence assays were also conducted to visualize whether S. suis enolase can specifically adhere to the surface of Hep-2 cells. Hep-2 cells cultured in 96-well cell plates were subjected to the customary procedure: washed 3 times with PBS (100 μL/well), fixated for 20 min with cold acetone (100 μL/well), and air-dried [35]. Either enolase (10 μg) or 1% bovine serum albumin (negative control) was added and incubated with the fixed Hep-2 cells at 37°C for 1 h. After the cultured cells were washed 3 more times with PBS, mouse antienolase serum was added, and the cells were incubated at 37°C for 30 min. Then the washed cells were incubated with goat anti-mouse IgG-fluorescein isothiocyanate (FITC) (Sigma) at 37°C for 30 min. Finally, the samples were washed 3 times and examined using a fluorescence microscope

Flow cytometry analysisFlow cytometry was applied to more narrowly determine the location of enolase on SS2 bacterial cells. Briefly, the overnight culture (108 colony-forming units/mL) was incubated with rabbit-antienolase serum. After 1 h, cells were washed and then incubated with goat anti-rabbit IgG-FITC for 1 h, which was followed by paraformaldehyde fixation for 30 min

Adherence assays of SS2 to Hep-2 cellsHep-2 cells were grown to confluence in 24-well tissue culture plates (0.5–1.0×106cells/well) and washed with Roswell Park Memorial Institute 1640 medium (RPMI 1640) without fetal calf serum and without antibiotics. Overnight culture of SS2 was adjusted to an optical density at 600 nm of 1.0. Hep-2 cells were cultured in 24-well plates to confluence, starved with RPMI 1640 without serum or antibiotics overnight, and infected with bacteria at a multiplicity of infection of 1:50 in RPMI 1640 in the presence of increasing amounts of free enolase. After 3 h of incubation at 37°C in 5% CO2, unbound bacteria were removed by means of PBS washing and treated with a lysis buffer (RPMI 1640 containing 0.1% trypsin and 1.0% Triton ×100). All experiments were performed in triplicate wells and repeated 3 times

Protection test of enolase in miceBALB/c mice (4 weeks old) were immunized subcutaneously at multiple sites with ∼25 μg of purified enolase emulsified 1:1 with Freund complete adjuvant. The mice received 1 booster injection after 14 d with the same antigen concentration emulsified 1:1 with Freund incomplete adjuvant, and after 7 d, the mice were immunized once again. After 1 week, the mouse model was used to test the protection efficacy of enolase against lethal infection with SS2 as described by Jacobs et al [36]. Mice were monitored and scored for survival for 7 d. All the experiments of animal infection were conducted in compliance with the regulations of the Nanjing Ethics Committee

Results

Expression and characterization of S. suisenolaseThe open reading frame of the S. suis gene eno is 1308 bp in length, encoding an enolase enzyme of 435 amino acid residues. Multiple sequence alignments showed that S. suis enolase is very similar to the homologues among other Streptococcus species (Figure 1). Six conserved residues were found in enolase proteins, 3 of which (D242, E291, and 318D) are critical for its interaction with its cofactor, manganese; the other 3 (164E, 205E, and 341L) are important for enzymatic activity (Figure 1). The secondary structure of enolase is proposed to consist of 15 α-helices and 16 β-sheets (Figure 1)

Figure 1

Multiple sequence alignments of Streptococcus suis enolase with related homologous proteins at the amino acid level. The enolase used here is from S. suis strain 05ZYH33 (FJ895346), Streptococcus mutants (ACG64169), and Streptococcus pneumonia (AAT86711). The image was generated using ESPript software, version 2.2, after the raw data were processed using ClustalW2. The protein secondary structure of enolase consists of 15 α-helices, 16 β-sheets, and 5 coils. α1, α-helix 1; α2, α-helix 2; α3, α-helix 3; …; β1, β-sheet 1; β2, β-sheet 2; β3, β-sheet 3; …; η1, coil 1; η2, coil 2; η3, coil 3; …; T, turn. The black triangles indicate the 3 conserved amino acid residues (D242, E291, and 318D) required for binding to the enzymatic cofactor, manganese. The asterisks indicate the remaining 3 conserved residues (164E, 205E, and 341L), which are recognized as enzymatic active sites. The known crystal structure of S. pneumonia enolase (Protein Data Bank entry 1W6T) was used as a reference [37]

Figure 1

Multiple sequence alignments of Streptococcus suis enolase with related homologous proteins at the amino acid level. The enolase used here is from S. suis strain 05ZYH33 (FJ895346), Streptococcus mutants (ACG64169), and Streptococcus pneumonia (AAT86711). The image was generated using ESPript software, version 2.2, after the raw data were processed using ClustalW2. The protein secondary structure of enolase consists of 15 α-helices, 16 β-sheets, and 5 coils. α1, α-helix 1; α2, α-helix 2; α3, α-helix 3; …; β1, β-sheet 1; β2, β-sheet 2; β3, β-sheet 3; …; η1, coil 1; η2, coil 2; η3, coil 3; …; T, turn. The black triangles indicate the 3 conserved amino acid residues (D242, E291, and 318D) required for binding to the enzymatic cofactor, manganese. The asterisks indicate the remaining 3 conserved residues (164E, 205E, and 341L), which are recognized as enzymatic active sites. The known crystal structure of S. pneumonia enolase (Protein Data Bank entry 1W6T) was used as a reference [37]

S. suis eno was engineered to produce an N-terminal 6×His-tag enolase protein in E. coli BL21 (DE3) so its biochemical properties could be documented. After 3 h of induction under the condition of 1 mmol/L IPTG, high-level expression of the recombinant protein was observed (>60 kDa), which accounts for ∼40% of the total protein (not shown). Subsequently, the protein was purified using affinity chromatography on a nickel column, followed by FPLC on a Superdex-200 column (Figure 2A). The purity of the recombinant enolase was verified by means of SDS-PAGE (Figure 2). An analytical FPLC assay clearly showed that the molecular mass of S. suis enolase is >440 kDa, which is ∼8 times the estimated formula size (Figure 2B and 2C). This observation can be verified mostly by structural evidence that S. pneumonia enolase is an octameric protein [37]. We used the method of structural modeling to determine the structure of S. suis enolase, which may be useful for molecule design in the development of medicine or therapeutics applied for the treatment of SS2 infections (Figure 2D and 2E). In addition, we propose a working model of S. suis enolase that may partially account for its biological role in triggering host immunity (Figure 2F)

Figure 2

Evidence that Streptococcus suis enolase is an octamer protein and models of its structure and function. A, Fast-phase liquid chromatography (FPLC) profile of S. suis enolase. The purified enolase was subjected to gel filtration on a Superdex 200HR 10/30 column (Amersham). The inset gel shows 2 samples (a and b) that were eluted at different volumes and then collected for visualization on 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis. B, Repeated FPLC analyses of enolase. The enolase from peak b in panel A was reloaded on a Superdex 200HR 10/30 column to estimate its molecular mass. A perfect peak (∼11 mL, representing 400–500 kDa) is verified here, in which the protein is of much purity (inside gel), with an approximate molecular weight of >66 kDa. Given the FPLC profile, the apparent molecular weight should vary from 400 to 500 kDa, implying that S. suis enolase behaves as an octamer. C, Estimate of the molecular weight of S. suis enolase (same as in panel B) using a standard curve. Standardized proteins (Pharmacia) were run on a Superdex 200HR 10/30 column to determine the relative molecular mass of enolase. The protein of interest is indicated by the arrow. According to the elution behavior of standard proteins, the molecular mass of S. suis enolase is >440 kDa, suggesting that it is an octamer. This is consistent with observations of the crystal structure of Streptococcus pneumonia enolase [37, 40]. D, E, Cartoon depiction and surface diagram, respectively, of S. suis enolase. Structural modeling was performed using CPHmodels, and the structure was generated using Visual Molecular Dynamics. Blue, coil; purple, α-helix; yellow, β-sheet; C, C-terminus of enolase; N, N-terminus of enolase. F, Proposed working model of S. suis enolase. S. suis enolase, in the form of an octamer, can be exported to cell surface and stimulate host humoral response in the process of infection. One column represents a monomer of enolase, and a bundle of 8 columns represents S. suis enolase in an octamer. mAU, milli-absorbance unit; Mw, molecular weight

Figure 2

Evidence that Streptococcus suis enolase is an octamer protein and models of its structure and function. A, Fast-phase liquid chromatography (FPLC) profile of S. suis enolase. The purified enolase was subjected to gel filtration on a Superdex 200HR 10/30 column (Amersham). The inset gel shows 2 samples (a and b) that were eluted at different volumes and then collected for visualization on 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis. B, Repeated FPLC analyses of enolase. The enolase from peak b in panel A was reloaded on a Superdex 200HR 10/30 column to estimate its molecular mass. A perfect peak (∼11 mL, representing 400–500 kDa) is verified here, in which the protein is of much purity (inside gel), with an approximate molecular weight of >66 kDa. Given the FPLC profile, the apparent molecular weight should vary from 400 to 500 kDa, implying that S. suis enolase behaves as an octamer. C, Estimate of the molecular weight of S. suis enolase (same as in panel B) using a standard curve. Standardized proteins (Pharmacia) were run on a Superdex 200HR 10/30 column to determine the relative molecular mass of enolase. The protein of interest is indicated by the arrow. According to the elution behavior of standard proteins, the molecular mass of S. suis enolase is >440 kDa, suggesting that it is an octamer. This is consistent with observations of the crystal structure of Streptococcus pneumonia enolase [37, 40]. D, E, Cartoon depiction and surface diagram, respectively, of S. suis enolase. Structural modeling was performed using CPHmodels, and the structure was generated using Visual Molecular Dynamics. Blue, coil; purple, α-helix; yellow, β-sheet; C, C-terminus of enolase; N, N-terminus of enolase. F, Proposed working model of S. suis enolase. S. suis enolase, in the form of an octamer, can be exported to cell surface and stimulate host humoral response in the process of infection. One column represents a monomer of enolase, and a bundle of 8 columns represents S. suis enolase in an octamer. mAU, milli-absorbance unit; Mw, molecular weight

Enzymatic activity ofS. suisenolaseIn general, enolase catalyzes the conversion of 2-PGE to PEP. After the concentration of the recombinant enolase was quantified at 280 nm, the enzymatic activity of enolase was measured spectrophotometrically at 240 nm. Michaelis-Menten kinetics of S. suis enolase validated the conversion of 2-PGE to PEP at 240 nm (Figure 3A). Then double-reciprocal Lineweaver-Burk plots were made to determine Vmax and Km for enolase (Figure 3B). The values of Km and Vmax were determined to be 2.47 mmol and 2.32 mmol/L/min, respectively (Figure 3B)

Figure 3

Enzymatic characterization of purified enolase. A, Michaelis-Menten kinetics of Streptococcus suis enolase. B, Determination of Vmax and Km for enolase by means of a Lineweaver-Burk plot (double-reciprocal plot). The values found were Vmax=1/0.4052 and Km=1/0.4308. The data were collected from 3 independent assays; shown here are the mean values ± standard deviation

Figure 3

Enzymatic characterization of purified enolase. A, Michaelis-Menten kinetics of Streptococcus suis enolase. B, Determination of Vmax and Km for enolase by means of a Lineweaver-Burk plot (double-reciprocal plot). The values found were Vmax=1/0.4052 and Km=1/0.4308. The data were collected from 3 independent assays; shown here are the mean values ± standard deviation

Immunogenicity ofS. suisenolaseA Western blot was performed to test the antigenicity and immunogenicity of S. suis enolase (Figure 4A). The result suggested that enolase can react strongly with convalescent-phase serum from pigs clinically infected with SS2 (Figure 4B). In addition, we also tested the specificity of our prepared antienolase polyclonal serum. As we expected, the Western blot results indicated that not only can the acquired antienolase antibody respond effectively to the purified recombinant enolase, but it also recognizes specifically the native enolase from SS2 crude extract (Figure 4C). In addition, we performed an enolase-based ELISA to evaluate its potential as a diagnostic antigen for investigation of S. suis infection (Figure 4D). Over 100 samples of suspicious pig serum were tested. Here, we show only 1 representative result (Figure 4D). The acquired data demonstrated that the ELISA can unambiguously distinguish between the positive, suspicious, and negative samples (Figure 4D). It seems that S. suis enolase possesses robust immunogenicity, which is a finding similar to our previous observations regarding Sao, a classical surface-anchored protein with a C-terminal LPXTG motif [33]

Figure 4

Immunogenicity of Streptococcus suis enolase sodium dodecyl sulphate polyacrylamide gel electrophoresis behavior (A) and Western blot analyses (B) of the purified S. suis enolase. The first antibody is convalescent-phase swine serum against S. suis strain 05ZYH33. C, Specificity of antienolase antibody. Lane 1, purified enolase; lane 2, lysated S. suis after sonication and lysozyme-mediated disruption; lane 3, negative control (the unrelated sample, Escherichia coli crude extract). Antienolase polyclonal serum is shown to recognize the native enolase in the lysate of S. suis. D, Development of an enolase-based ELISA method for monitoring S. suis infection. Samples 1–20 are representative serum samples collected from pig farms. Plus sign, positive serum sample confirmed in the laboratory; minus sign, negative serum sample confirmed in the laboratory. Two baselines, 1 positive and 1 negative, are indicated in dark gray and light gray, respectively. The samples with ELISA scores between the 2 baselines are considered to be suspicious

Figure 4

Immunogenicity of Streptococcus suis enolase sodium dodecyl sulphate polyacrylamide gel electrophoresis behavior (A) and Western blot analyses (B) of the purified S. suis enolase. The first antibody is convalescent-phase swine serum against S. suis strain 05ZYH33. C, Specificity of antienolase antibody. Lane 1, purified enolase; lane 2, lysated S. suis after sonication and lysozyme-mediated disruption; lane 3, negative control (the unrelated sample, Escherichia coli crude extract). Antienolase polyclonal serum is shown to recognize the native enolase in the lysate of S. suis. D, Development of an enolase-based ELISA method for monitoring S. suis infection. Samples 1–20 are representative serum samples collected from pig farms. Plus sign, positive serum sample confirmed in the laboratory; minus sign, negative serum sample confirmed in the laboratory. Two baselines, 1 positive and 1 negative, are indicated in dark gray and light gray, respectively. The samples with ELISA scores between the 2 baselines are considered to be suspicious

Surface display ofS. suisenolaseTo investigate the distribution of enolase in S. suis, 3 independent lines of approach were used. First, ELISA showed that samples from 2 locations (the cell wall and the cytoplasm) can both produce higher antiserum titers against postimmune serum than against preimmune serum (Figure 5A). The immunological data showed that enolase can be displayed on the SS2 surface and effectively induce the humoral immunity of its host, piglets. To test the hypothesis that some immunodominant B cell epitopes could be present in enolase, we attempted to perform a bioinformatics-based investigation to resolve this issue (data not shown). Although it is not certain thus far that T cell immunity is involved in the interaction of SS2 enolase with the host (piglets), we still compiled a list of MHC II-restricted peptides that have potential applications (data not shown). Then fluorescence-activated cell sorting (FACS) analysis was applied to determine the subcellular location of enolase. We found that the unlabeled SS2 is distributed normally (Figure 5B) with a mean fluorescence intensity (MFI) close to that of the labeled SS2 treated with rabbit preimmune serum (Figure 5C and 5E). However, the MFI of bacteria treated with postimmune serum was twice that of bacteria treated with preimmune serum (Figure 5D). The reason lies in the presence of the enolase antigen both in the cytoplasm and on the bacterial cell surface, which is well recognized by rabbit antienolase antibody (in postimmune serum). In addition, the preliminary results of our immunogold electron microscopy analysis support the above-mentioned general conclusion made on the basis of the results of our fractionation and FACS analyses (data not shown) that S. suis enolase antigen could indeed be exported to the SS2 bacterial cell surface

Figure 5

Surface display of Streptococcus suis enolase. A, ELISA-based subcellular localization of enolase in S. suis serotype 2, showing antibodies before (white) and after (gray) immunization. B-E, Extracellular detection of S. suis enolase by fluorescence-activated cell sorting (FACS) analysis, which suggests that the unlabeled bacteria (B) can form a group (ie, the sample is suitable for localization by means of a FACS detector). C, Mean fluorescence intensity (MFI) of the unlabeled bacteria. D, E, MFI of bacteria treated with rabbit antienolase serum and of bacteria treated with rabbit preimmune serum, respectively

Figure 5

Surface display of Streptococcus suis enolase. A, ELISA-based subcellular localization of enolase in S. suis serotype 2, showing antibodies before (white) and after (gray) immunization. B-E, Extracellular detection of S. suis enolase by fluorescence-activated cell sorting (FACS) analysis, which suggests that the unlabeled bacteria (B) can form a group (ie, the sample is suitable for localization by means of a FACS detector). C, Mean fluorescence intensity (MFI) of the unlabeled bacteria. D, E, MFI of bacteria treated with rabbit antienolase serum and of bacteria treated with rabbit preimmune serum, respectively

The involvement of enolase in SS2 adherenceImmunofluorescence analyses were conducted to test whether SS2 enolase contributes to SS2 adherence. We found much evidence of enolase binding to the SS2 surface (Figure 6A). We also examined SS2 adherence to Hep-2 cells in the presence of increasing amounts of free purified enolase and found that SS2 adherence to Hep-2 cells was inhibited by enolase in a dose-dependent manner (Figure 6B). When the concentration of enolase reached 10 μg/well, ∼50% of SS2 adherence was inhibited. Therefore, we conclude that an interaction between SS2 enolase and Hep-2 cells plays a critical role in the bacterial adherence process. However, studies of its possible role in SS2 virulence have been hampered by the unavailability of the knockout mutant of eno, which is an essential gene for Streptococcus

Figure 6

Role of Streptococcus suis enolase in adherence. A, Immunofluorescence analyses showing binding of the purified enolase to Hep-2 cells. Blue indicates the Hep-2 cell nucleus, and green indicates enolase bound to the surface of the Hep-2 cell. B, Role of S. suis serotype 2 (SS2) enolase in its adherence to Hep-2 cells. The SS2 associated with Hep-2 cells were counted as colony-forming units (CFUs). Dose-dependent inhibition of SS2 adherence to Hep-2 cells was determined in the presence of increasing amounts of purified enolase

Figure 6

Role of Streptococcus suis enolase in adherence. A, Immunofluorescence analyses showing binding of the purified enolase to Hep-2 cells. Blue indicates the Hep-2 cell nucleus, and green indicates enolase bound to the surface of the Hep-2 cell. B, Role of S. suis serotype 2 (SS2) enolase in its adherence to Hep-2 cells. The SS2 associated with Hep-2 cells were counted as colony-forming units (CFUs). Dose-dependent inhibition of SS2 adherence to Hep-2 cells was determined in the presence of increasing amounts of purified enolase

Protection conferred by enolase to mice infected with SS2We assessed the protection conferred by enolase to mice that were infected with SS2. Two days after inoculation, 15 of 15 mice in the negative control group had died, but the mice immunized with suilysin or enolase survived and did not show any clinical signs of SS2 infection (eg, ruffled hair, absentmindedness, and poor appetite) (Table 1). Therefore, we conclude that, for mice, enolase can confer a level of protection similar to that of suilysin, a well-known protective antigen [36, 38]. To better understand the molecular mechanism by which enolase functions as a protective antigen, we used a bioinformatics approach to dissect possible B cell epitopes and MHC II-restricted peptides of enolase (data not shown). Moreover, we propose a working model of S. suis enolase that describes the pathway for an atypical export of the octameric enolase and induction of the host immunological response (Figure 2F). This proposed model should help us understand the essence of enolase as a protective antigen displayed on the bacterial cell surface

Table 1

Protection Efficacy of Purified Enolase against Streptococcus suis Serotype 2 Infection

Table 1

Protection Efficacy of Purified Enolase against Streptococcus suis Serotype 2 Infection

Discussion

In this study, we investigated S. suis enolase, which has a high similarity to the homologues in the other Streptococcus species. Enzymatic test results showed that S. suis enolase that is overexpressed in vitro is a functional member. Although Esgleas et al [31] recently reported that α-enolase of S. suis is a fibronectin-binding protein, data from our 3 lines of investigation (immunoelctron microscopy, FACS, and ELISA) provide evidence that S. suis enolase occurs on the bacterial cell surface, as well as in the cytoplasm. It is not exported to the cell surface via the known mechanism that is generally associated with proteins that have the anchor motif LPXTG. In retrospect, there have been similar cases of anchorless enzymes being present on the cell surface and executing an infection-related function. Therefore, we speculated that the presence of S. suis enolase on the cell surface could be correlated with high invasiveness of Chinese SS2 strains

Immunological data demonstrated that enolase exhibits strong immunogenicity. On the basis of our previous experience with a surface antigen protein (Sao) [33], we developed an effective enolase-based ELISA method for monitoring SS2 infection in pigs, which implies that enolase shows great promise as a diagnostic antigen. To better address its possible role in the early events of SS2 infection, we conducted cell line- and mouse model-based exploration. The presence of enolase on Hep-2 cells was observed to greatly reduce SS2 adhesion, which indicates that SS2 enolase contributes to the bacterial adhesion to host surface. In fact, recent reports suggested that the ability of other pathogenic bacteria to express plasminogen-binding proteins, such as enolase, may be an important determinant of virulence [37, 39]. It is of much interest that SS2 infection can trigger the production of antienolase antibodies in swine. It is hoped that enolase can produce strong protective immunity against SS2 infection. As we expected, we found that immunization with enolase confers protection against lethal SS2 infection in mice, prompting us to believe it could potentially be a candidate subunit vaccine against infection by the bacteria [18, 19]. Very recently, a similar finding was reported regarding Streptococcus sobrinus, which indicated that oral therapeutic vaccination with recombinant enolase confers protection against dental caries in rats [30]. The findings that facilitated the design of a highly effective epitope vaccine prompted us to further address B cell epitopes and MHC II-restricted peptides for SS2 enolase (data not shown). Additionally, we also constructed a model of the structure of S. suis enolase, which may be useful for molecule design in developing medicine or therapeutics applied for the treatment of SS2 infections (Figure 2D and 2E)

In summary, we showed evidence for the presence of S. suis enolase on the bacterial cell surface. We also used the strong immunogenicity of enolase to develop an enolase-based ELISA diagnostic method and demonstrated that it exhibits potential as an effective subunit vaccine against SS2 infections

Acknowledgments

We thank the Deputy Editor, David C. Hooper, and the Associate Editor, Frederick Southwick, for their helpful comments and the anonymous reviewers for their constructive suggestions on our manuscript

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Potential conflicts of interest: none reported
Financial support: National High-Tech Research and Development (project 863, grant 2006AA0Z455); National Key Technologies Research and Development Program (grants 2006BAD06A01 and 2006BAD06A04); National Basic Research Program of China (program 973, grant 2005CB523001); National Natural Science Foundation of China (grants 30600533, 30670105, 30671848, 30730081, and 30972638); Natural Science Foundation of Jiangsu Province (grants BK2007013, BK2008066, and BK2009042); Foundation of Innovation of Medical Science and Technology (grant 07Z045); “122” Project of Talent Cultivating in Health Professions

Author notes

a
Present affiliations: Dept of Microbiology, University of Illinois, Urbana, Illinois (Y.F.); Yangzhou Vocational College of Environment and Resources, Yangzhou, Jiangsu, China (W.S.)
b
Y.F., X.P., and W.S. contributed equally to this article