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Streptococcus suis has been described as the etiological agent for a number of infectious disease syndromes in pigs, including arthritis, septicemia, meningitis, and pneumonia. S. suis produces a secreted hemolysin (suilysin) which has been suggested as playing a role in virulence (2, 4-7, 10). In order to investigate the role played by suilysin in the pathogenesis of a European serotype 2 isolate of S. suis, we generated a defined allelic-replacement mutant of the sly gene and compared the wild-type organism with the sly mutant in a number of assays.

Bacterial strains and media. S. suis type 2 strain P1/7 was grown on Columbia agar (Oxoid) containing 10% defibrinated horse blood or in liquid cultures of Todd-Hewitt broth (Oxoid) supplemented with 7% fetal calf serum (FCS) (Gibco). Allelic-replacement mutants of S. suis were maintained on 1 µg of erythromycin (Sigma) per ml.

Mutagenesis of the suilysin gene from S. suis type 2. An erythromycin resistance gene cassette was introduced into an EcoRV site within a 1,278-bp fragment of the sly gene, contained in the vector pT7-Blue (Stratagene), amplified by PCR using primers suis1 (5'-AGCTTGACTTACGAGCCACAAGAG-3') and suis2 (5'-CCACCATTCCCAAGCTAATCCTGT-3') with chromosomal DNA from P1/7 as a template. The resulting plasmid, pSUI-erm, contains the erm gene in the same orientation as the sly gene. Plasmid pSUI-erm was introduced into P1/7 by electroporation (22.5 V/cm, 25 µF, and 1,000 ). A transformant which had undergone a double-crossover event, confirmed by Southern hybridization and PCR, with concomitant insertion mutation of the sly gene, was isolated and named S7c.

Phenotypic analysis of S7c. Overnight growth of suilysin mutant S7c on Columbia blood agar plates revealed no -hemolysis. Secreted proteins from anaerobically grown overnight cultures of P1/7 and S7c were concentrated 100-fold by ammonium sulfate (50%, wt/vol) precipitation; then 10 µl each of these preparations was spotted onto a Columbia horse blood agar plate and incubated at 37°C for 30 min. The proteins from P1/7 show clear zones of hemolysis, whereas there is a complete absence of hemolytic activity in the proteins obtained from S7c (Fig. 1a). The hemolytic activity from the wild-type parental bacteria was enhanced by the addition of -mercaptoethanol, as previously reported (7) (Fig. 1a).

View larger version (43K): [in this window] [in a new window]   FIG. 1.   Characterization of P1/7 and S7c culture supernatants. (a) Overnight culture supernatants from wild type P1/7 (wt) and S7c (S7c) were concentrated 100-fold by ammonium sulfate precipitation, and 10-µl samples, indicated by circles, were overlaid onto a 7% (vol/vol) horse blood agar plate, followed by incubation at 37°C for 60 min. Samples in track B were treated with -mercaptoethanol to a final concentration of 1 mM. PBS, phosphate-buffered saline. (b) Western blot of culture supernatants from P1/7 and S7c using antisuilysin monoclonal antibody. Proteins secreted from overnight culture supernatants from P1/7 (track A) and S7c (track B) were concentrated 25 times, separated on a polyacrylamide gel, and Western blotted onto nitrocellulose, followed by development with a monoclonal antibody specific for suilysin. The band corresponding to suilysin in track A is completely absent from track B. Molecular weights are in thousands.

Cell culture and cytotoxicity assay. Murine macrophage-like J774.2 cells were split into 96-well plates (approximately 1.5 × 10⁵ cells/well) and maintained in Dulbecco modified Eagle medium supplemented with 3% FCS and 2 mM glutamine (assay buffer). Bacterial inocula (5 × 10⁷ CFU per 100 µl, in assay buffer), generated from overnight cultures of P1/7 and S7c, were added to three experimental wells and incubated at 37°C with 5% carbon dioxide for 3 and 5 h. Relative cytotoxicity was assayed as lactate dehydrogenase (LDH) release as determined by the CytoTox96 kit (Promega). The experiment was repeated on three separate occasions (Fig. 2). The parental strain, P1/7, caused extensive damage to cell monolayers in a time-dependent manner. In comparison, S7c elicited only approximately 4% of maximal LDH release after 5 h of incubation. Bacterial viable counts during the experiment revealed identical numbers of CFU for P1/7 and S7c. These experiments show that S. suis efficiently kills J774.2 cells and that this effect is dependent on the presence of suilysin, which is thus probably the only cytolysin produced by S. suis.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Comparison of cytotoxicities of P1/7 and S7c for J774.2 cells. Monolayers of J774. 2 cells (2 × 105 cells) were infected with P1/7 (P) or S7c (S). Relative levels of LDH released at 3 and 5 h were measured and plotted as percentages of the maximum lysis achieved by chemical treatment. Experiments were performed in triplicate for each datum point. Where no bars are present, the lysis was effectively zero. Spontaneous lysis (C) occurred without the addition of bacteria. It is clear that wild-type bacteria cause substantial lysis of the cell monolayer, whereas the mutant lacking suilysin is unable to lyse the cells.

Mouse infection experiments. Overnight cultures of P1/7 and S7c, grown in Todd-Hewitt broth containing 10% FCS, were diluted in the same medium to achieve 10⁸, 10⁷, 10⁶, and 10⁵ CFU/0.5-ml dose. These 0.5-ml inocula were introduced intraperitoneally into 5-week-old female BALB/c mice, in groups of five mice per inoculum. Mice were monitored over a period of 1 week, during which deaths were recorded and moribund animals were humanely killed. Inoculation of 10⁸ and 10⁷ wild-type bacteria killed 9 of 10 mice and 8 of 10 mice, respectively, over the two experiments, within 48 h, providing 50% lethal doses (9) of log 6.45 and log 6.73, respectively, whereas all mice infected with S7c survived. These data show unequivocally that mutation of suilysin prevents S. suis from killing mice via the intraperitoneal route of infection.

Pig infection experiment. Pigs, aged between 35 and 40 days, were obtained from Cotswold Pigs Ltd. (Colsterworth Farm, United Kingdom). Fourteen days prior to challenge, animals were screened for the presence of major pathogens and injected with ceftiofur (Excenel; Pharmacia & Upjohn Ltd.) for three consecutive days. Bacteria for challenge were grown overnight in brain heart infusion broth at 37°C and were subcultured in the same medium for 6 h at 37°C. Cultures were diluted in phosphate-buffered saline (pH 7.2), and each animal was challenged by intravenous injection of 1 ml of phosphate-buffered saline containing between 1 × 10⁶ and 5 × 10⁶ CFU of either strain S7c (group 1) or P1/7 (group 2). Animals were monitored for clinical signs of disease every 3 h; piglets showing marked signs of illness were killed for postmortem examination. Surviving animals were killed for postmortem examination 44 h postinfection. At postmortem, a visual inspection was made in order to detect pathological lesions and swabs were taken from heart blood, the lateral ventricle of the brain, the lungs, the serous cavity, and two limbs for bacterial examination. Results (lesion scores, bacteriology, survival times) between groups were analyzed statistically using Student's t test via MINITAB release 12.1 for Windows. Tests were performed assuming equal variance and were repeated assuming unequal variance where necessary.