The gene encoding suilysin was cloned from Streptococcus suis serotype 2 strain P1/7. Analysis of the nucleotide and translated amino acid sequence confirmed suilysin to be a member of the thiol activated cytolysin group (TACY). The pneumolysin from Streptococcus pneumoniae is the most closely related orthologous gene known. Suilysin was overexpressed in E. coli in an active haemolytic form. A strong correlation between the presence of the sly gene and haemolytic activity in the supernatant of S. suis field strains was found. Of 158 strains tested, 63% contained the gene. Within the (most prevalent) serotype 2, the sly gene was demonstrated in 95% of the strains isolated in Eurasia, but only in 7% of the strains from North America.
Streptococcus suis is the causative agent of a contagious porcine disease, characterised by pneumonia, arthritis, septicaemia, pericarditis, meningitis and/or polyserositis [1, 2]. It also has a zoonotic character and can be isolated from a wide variety of mammals, including man . Thiol-activated cytolysins (TACY) are a family of pore-forming toxins produced by members of the Streptococcus, Listeria, Bacillus, and Clostridium genera . Recently, a new TACY was isolated from Streptococcus suis and designated suilysin . In contrast to other extracellular antigens, suilysin can protect pigs from virulent challenge after vaccination . In the present study the gene encoding suilysin was cloned and analysed. Furthermore, the prevalence of this gene among field strains was investigated.
2 Materials and methods
2.1 Bacterial strains and plasmids
S. suis strains P1/7 and B10 have been described . Other field strains and/or their DNA were kindly provided by Dr. Bjarne Nielsen (Intervet Scandinavia, Copenhagen, Denmark), Dr. Marcello Gottschalk (Université de Montréal, Canada), Dr. Hilde Smith (Institute for Animal Science and Health, Lelystad, The Netherlands), or Dr. Doug Burkhardt (Ambico, Dallas Center, IA, USA). E. coli host strain DH5a (genotype F−Φ80d lacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 hsdR17(r−K12 m+K12) deoR supE44 thi-1 λ-gyrA96 relA1) was purchased from Clontech (Palo Alto, Ca). E. coli host strain HMS174(DE3) with pLysS (genotype: F−recA r−K12 m+K12 Rifr (DE3) pLysS Cmr) and plasmid pET9c were purchased from Novagen (Madison, Wi.). The plasmid vector pCRtmII in the TA-cloning kit was purchased from Invitrogen (San Diego, CA, USA). Oligonucleotides used: IVRSE26 (5′-GGAGAGCTCATATGAARCARGAYATHAAYCARTAYTTYCA-3′); IVRSE27 (5′-CGGGATCCTTACCACCATTCCCAAGC-3′); IVRSE41 (5′-AACATACCAGTTGTTGCTGGCGGA); IVRSE42 (5′-CTGACCTCACACTGGAGTGAAACCA); Sly-F (5′-GGGAATTCCATATGAGAAAAAGTTCGCACTTGATTTT) and Sly-R (5′-CGGGATCCTTACTCTATCACCTCATCCGCATACT), were ordered from Pharmacia Biotech., Roosendaal, The Netherlands).
2.2 DNA techniques
All routine DNA manipulations were performed as described by Sambrook et al. . Plasmid DNA was isolated using Qiagen columns (Qiagen, Chatsworth, CA, USA) according to the manufacturer's directions. DNA fragments were isolated from agarose gels using the Geneclean II kit (BIO101, La Jolla, CA, USA). E. coli competent cells were made according to ‘Protocol 2 for frozen storage of competent cells’.
The Genome Walking Kit (Clontech, Palo Alto, CA, USA) was used to determine the nucleotide sequences of the complete sly gene and the adjacent regions. Briefly, chromosomal DNA of S. suis strain P1/7 was digested with PvuII or HincII and ligated to linkers according to the manufacturers' directions. Using oligonucleotides IVRSE41 or IVRSE42 in combination with primer AP1 from the Genome Walking Kit, fragments could be amplified and sequenced. The 1.3-kb NdeI/BamHI insert from pSly-1 was excised, labelled using an ECL direct labelling kit (Amersham Pharmacia Biotech Benelux, Roosendaal, The Netherlands) and used as a probe. Hybridisation was performed according to the manufacturers' directions.
2.3 Overexpression of suilysin in E. coli
On the basis of the sequence obtained after genome walking, primers Sly-F and Sly-R were designed for amplification and cloning of the sly gene (after NdeI/BamHI digestion of the PCR fragment) in expression vector pET9c, digested with the same enzymes. The resulting plasmid pSly-3 was transferred to expression strain HMS174(DE3) containing the compatible plasmid pLysS for stability. Cultures were grown in Terrific Broth , supplemented with 25 μg/ml of chloramphenicol and 50 μg/ml of kanamycin up to an OD600 of 0.7 and isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a concentration of 0.5 mM, followed by further growth for 3 h.
2.4 Haemolysin assay on blood plates
A volume of 10 μl of freshly grown E. coli culture in Luria-Bertani broth was spotted onto agar plates containing 6% (v/v) sheep red blood cells and 0.01 mM IPTG. Haemolysis was scored by visual inspection after incubation for 16 h at 37°C.
2.5 Titration of haemolytic activity
S. suis strains were inoculated on blood agar plates and grown for 24 h at 37°C in an atmosphere of 5% CO2. From these plates, 50 ml of Todd-Hewitt broth was inoculated and grown for 5 h at 37°C. The cells were removed by centrifugation for 15 min at 10 000×g and β-mercaptoethanol was added to the supernatant up to 0.1% (v/v). Titration of the hemolytic activity was performed as described previously .
2.6 Chromosomal DNA isolation
Chromosomal DNA from S. suis was isolated from the cell pellet from 50 ml of S. suis culture, grown in Todd-Hewitt broth. The pellet was suspended in 10 ml TEG , with 500 μg/ml of lysozyme and incubated 1 h on melting ice. Subsequently, 100 U mutanolysin was added and the mixture was incubated for 2 h at 37°C. After the addition of 1.1 ml of 10% (w/v) SDS, DNA isolation was performed as described .
2.7 PCR amplification
PCR amplification was performed using a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, USA). The PCR mixtures (100 μl each) contained 1 U SuperTaq, 1× SuperTaq buffer (HT Biotechnology, UK), 200 μM of dATP, dGTP, dCTP and dTTP, 10 pmol of oligos Sly-F and Sly-R and approximately 0.1 μg of chromosomal DNA as template. As the PCR program, the mixture was denatured for 2 min at 94°C. Subsequently, 30 cycles consisting of 30 s denaturation at 94°C; 1 min annealing at 53°C; and 3 min of elongation at 67°C. From each amplification 10 μl was tested on an agarose gel.
2.8 Nucleotide sequence analysis
The nucleotide sequence was determined by Baseclear (Leiden, The Netherlands), using a LI-COR Model 4000 Automatic Sequencer. Homologous nucleotide sequences from the TACY family were extracted from the GenBank, EMBL and/or Prosite databases (TACY signature database nr. PS00436). Multiple alignments were performed using ClustalX  and for phylogenetic analysis the Molecular Evolutionary Genetics Analysis (MEGA v.1.01; Sudhir Kumar, Koichiro Tamura and Msatoshi Nei, Pennsylvania State University, PA 16802, USA) was used.
Murine monoclonal antibodies against suilysin, isolated from S. suis strain P1/7 were produced according to standard procedures . Monoclonal antibody INT STS 01-29-1 was selected after reaction with suilysin in a Western blot experiment. Immunoglobulins were isolated from culture supernatant and conjugated to horseradish peroxidase as described . Polyclonal rabbit serum R2089, raised against suilysin, has been described .
3.1 Cloning and analysis of the sly gene
Based on the N-terminal amino acid sequence  and the consensus motif ECTGLAWWEW of the thiol activated cytolysin family , degenerated oligonucleotides IVRSE26 and IVRSE27 were designed by reversed translation. Using these primers, a DNA fragment of approximately 1.3 kb was amplified using chromosomal DNA from strain P1/7 as a template. After cloning into vector pCRII (the resulting plasmid was designated pSly-1), the sequence of the insert was determined to be homologous to other thiol-activated cytolysins. The remainder of the gene and some of the flanking regions were sequenced on PCR products obtained after genome walking. The complete open reading frame (ORF) was subsequently amplified using primers Sly-F and Sly-R and cloned as an NdeI/BamHI fragment into the expression vector pET9c. This plasmid was designated pSly-3. The sequencing data on the inserts of pSly-1, pSly-3, as well as all the sequencing data from the genome walking experiments were combined and submitted to the EMBL database (accession nr. Z36907). An ORF of 497 amino acid residues could be identified with a calculated molecular mass of 54 850. The first 27 amino acid residues fit the description of a signal peptide, since a positively charged N-terminal region is followed by a hydrophobic central region, and a more polar C-terminal region precedes the start of the mature protein. Moreover, the −1, −3 rule at the cleavage site applies . The N-terminal amino acid sequence as previously published  could be identified at residues 28–43 of rhis ORF. Sequence comparison with other members of the TACY family reveals amino acid sequence identities between 52% (for pneumolysin) and 38% (for alveolysin).
The ECTGLAWEWWT undecapeptide motif, characteristic of the TACY-family , is completely conserved. The two regions which are present in other TACY, with homology to the C-reactive protein , are also present in suilysin (from residues 285 to 325 and from 396 to 425, resp.).
3.2 Phylogenetic analysis
Complete TACY-protein sequences were aligned and analysed by UPGMA and Neighbor-joining methods using various parameters. The basic topology of all resulting trees (with minor differences in branch length) was as in Fig. 1. Phylogenetically, pneumolysin is the closest relative.
Phylogenetic analysis of protein sequences showing the relationship of suilysin to the other TACYs. Displayed is an unrooted dendrograph of complete TACY amino acid sequences from various species. Accession numbers are: Arcanobacterium pyogenes (U84782); S. pneumoniae (P11990); S. suis (Z36907); Listeria ivanovii (P31831); L. monocytogenes (P13128); L. seeligeri (P31830); Bacillus alvei (P23564); C. perfringens (P19995); S. pyogenes (P21131); S. canis (D16825) and S. equisimilis (D16824). Sequences were aligned using the ClustalX program and phylogenetically analysed by Neighbor Joining algorithms.
3.3 Overexpression of suilysin in E. coli
The expression plasmid pSly-3 was transferred to the E. coli HMS174(DE3) host strain containing the compatible plasmid pLysS. After induction with IPTG, a band with an approximate molecular mass of 58 kDa was observed after analysis on a Coomassie Brilliant Blue stained polyacrylamide gel (Fig. 2A). Both the suilysin isolated from S. suis (lane 1) and the recombinant suilysin isolated from E. coli (lane 3) migrate at the same location in the gel. This band was shown to react with specific polyclonal rabbit serum as well as monoclonal antibody (Fig. 2B and C) raised against suilysin isolated from S. suis. In addition, the suilysin expressed in E. coli was shown to have haemolytic activity (see Fig. 3).
Overexpression of suilysin in E. coli HMS174(DE3) containing pLysS. Whole cell lysates from IPTG induced cultures were analysed on polyacrylamide gel and stained with Coomassie Brilliant Blue (A) or blotted onto membrane and reacted with polyclonal antibody R2089 (B) or monoclonal antibody INT STS01-29-1 (C). Lanes 1 contain suilysin isolated from S. suis strain P1/7, lanes 2 contain E. coli lysates with empty vector pET9c, and lanes 3 contain E. coli lysates with pSly-3. The lane containing the marker proteins is indicated with an M, and apparent molecular mass is indicated on the left side in kDa.
Haemolysin plate assay. Blood plate (6% sheep red blood cells) containing IPTG, inoculated with HMS174(DE3)+pLysS, containing either the empty vector pET9c (on the left side) or pSly-3 (on the right side).
3.4 Presence of the sly gene among field strains
The presence of the sly gene was demonstrated by PCR, using primers Sly-F and Sly-R in 100 out of 158 field strains (Table 1). In 13 strains, negative in the PCR, the absence of the gene was confirmed in a Southern blotting experiment using the sly gene as a probe (results not shown). With respect to the geographical origin of isolation, the sly gene is present in 95% of the serotype 2 strains from Europe and Asia, but only in 7% from the strains from North America. A number of the strains tested were previously grouped according to their degree of virulence for pigs . All (highly) virulent strains (7 isolates) or strains isolated from the central nervous system (5 isolates) were shown to contain the sly gene. Of the 8 avirulent strains, 6 were shown to contain the sly gene. No correlation between the presence of muramidase released protein (MRP) and/or extracellular protein factor (EF), and the presence of the sly gene was found.
Presence of the sly gene in S. suis field strains as determined by PCR amplification
The number of strains containing the sly gene is indicated, followed by the number of strains tested.
3.5 Expression of haemolytic activity
For a total of 62 strains, the haemolytic activity in the culture supernatant was quantified and titers were calculated as the reciprocal value of the highest dilution inducing at least 50% lysis of erythrocytes. Values between 0 and 8 were found. All measurements were performed either in duplo or triplo and average values were calculated. A haemolysin titer of 1 or higher was regarded as indicative of the presence of haemolysin. In 29 strains both the presence of the gene and haemolytic activity in the supernatant was demonstrated. The average haemolysin titer was 4.0. In 28 strains both the gene and haemolytic activity were absent. In four strains with low titers (between 1 and 2) the presence of the gene could not be demonstrated, and one strain was shown to have the gene but not the haemolytic activity. Correlation between the presence and absence of both the sly gene and the haemolytic activity was shown to be significant (P<0.05 by Pearson's chi-square).
The sequence analysis of the sly gene and its translation product are in agreement with the published amino acid sequence and biochemical data and confirm that it is a member of the TACY family. The consensus sequence ECTGLAWEWWR, important for haemolytic activity, is perfectly conserved, and the two regions implicated in complement activation in pneumolysin, are present in suilysin as well. However, in the second region suilysin contains Asn413, whereas other members of the TACY family have an Asp residue at this position. When this Asp is mutated into an Asn, pneumolysin looses most of its complement activation activity . So far, it is not known whether suilysin actually binds porcine immunoglobulin.
Although suilysin has the highest sequence identity and also the closest phylogenetic relationship with pneumolysin there are some striking differences. Firstly, pneumolysin is an intracellular protein, whereas suilysin is exported . This is supported by the sequence data, since a putative signal peptide precedes the experimentally determined start of the mature protein. Secondly, pneumolysin is expressed by virtually every clinical isolate of S. pneumoniae, whereas the suilysin is absent in about 37% of all S. suis field strains tested. A number of TACY has been proven to be virulence factors , and vaccination experiments indicate that suilysin is one as well . However, since most isolates were obtained from diseased pigs, S. suis apparently has more important and/or other virulence factors to compensate for the absence of suilysin. The available clinical data on the field isolates are not sufficient to link the presence of suilysin to specific clinical symptoms or to increased virulence.
Although suilysin is able to afford protection across the serotypes , the absence of the toxin in quite a number of field isolates implies that other vaccine components will be necessary for protection against all field strains.
We thank P. Loeffen for excellent assistance with the Western blotting experiments, and all the people mentioned in Section 2 for sending us the S. suis field strains or derived DNA.