The cell envelope subtilisin-like proteinase is a virulence determinant for Streptococcus suis

Abstract

Background: Streptococcus suis is a major swine pathogen and zoonotic agent that mainly causes septicemia, meningitis, and endocarditis. It has recently been suggested that proteinases produced by S. suis (serotype 2) are potential virulence determinants. In the present study, we screened a S. suis mutant library created by the insertion of Tn917 transposon in order to isolate a mutant deficient in a cell surface proteinase. We characterized the gene and assessed the proteinase for its potential as a virulence factor.

Results: Two mutants (G6G and M3G) possessing a single Tn917 insertion were isolated. The affected gene coded for a protein (SSU0757) that shared a high degree of identity with Streptococccus thermophilus PrtS (95.9%) and, to a lesser extent, with Streptococcus agalactiae CspA (49.5%), which are cell surface serine proteinases. The SSU0757 protein had a calculated molecular mass of 169.6 kDa and contained the catalytic triad characteristic of subtilisin family proteinases: motif I (Asp200), motif II (His239), and motif III (Ser568). SSU0757 also had the Gram-positive cell wall anchoring motif (Leu-Pro-X-Thr-Gly) at the carboxy-terminus, which was followed by a hydrophobic domain. All the S. suis isolates tested, which belonged to different serotypes, possessed the gene encoding the SSU0757 protein. The two mutants devoid of subtilisin-like proteinase activity had longer generation times and were more susceptible to killing by whole blood than the wild-type parent strain P1/7. The virulence of the G6G and M3G mutants was compared to the wild-type strain in the CD1 mouse model. Significant differences in mortality rates were noted between the P1/7 group and the M3G and G6G groups (p < 0.001).

Conclusion: In summary, we identified a gene coding for a cell surface subtilisin-like serine proteinase that is widely distributed in S. suis. Evidences were brought for the involvement of this proteinase in S. suis virulence.

Figure 1

Southern blot of S. suis P1/7 and the Tn917 mutants. Chromosomal DNA was digested with HindIII restriction endonuclease and hybridized with a DIG-labeled probe specific for the erm gene. Lane 1, wild-type parent strain P1/7; lane 2, mutant J9G; lane 3, mutant M3G; lane 4, G6G.

Figure 2

Alignment of the catalytic triad (Asp₂₀₀ - His₂₃₉ - Ser₅₆₈; indicated by arrows) of S. suis SSU057 and homologous streptococcal subtilisin-like proteinases. Each catalytic triad is identified by UniProtKB accession numbers: A4VUI8 + A4VUI9 correspond to S. suis 05ZYH33 (SSU05-0811 + SSU05-0812); A4WOT0 + A4WOT1 correspond to S. suis 98HAH33 (SSU98-0811 + SSU98-0812); Q9F8Q4 corresponds to S. thermophilus PrtS; Q3JYS0 corresponds to S. agalactiae CspA; A3CQ08 corresponds to S. sanguinis PrtS; Q9A180 corresponds to S. pyogenes PrtS; Q3HV58 corresponds to S. pyogenes ScpC; P15926 corresponds to S. pyogenes ScpA; Q3K0M1 corresponds to S. agalactiae ScpB; Q04LP0 corresponds to S. pneumoniae PrtA.

Figure 3

Distribution of the gene coding for the SSU0757 protein in various S. suis strains. Lane 1, DNA molecular weight markers; lane 2, S428 (serotype 1); lane 3, P1/7 (serotype 2); lane 4, 90-1330 (serotype 2); lane 5, S735 (serotype 2); lane 6, 65 (serotype 2); lane 7, 31533 (serotype 2); lane 8, 89-4223 (serotype 2); lane 9, 89-999 (serotype 2); lane 10, 2651 (serotype 1/2); lane 11, 4961 (serotype 3); lane 12, Amy12C (serotype 5); lane 13, 1078212 (untypeable); lane 14, 1079277 (untypeable).

Figure 4

Survival of S. suis wild-type strain P1/7 and mutants M3G and G6G in human whole blood. Mixtures were incubated at 37°C for 4 h. A value of 100% was given to the colony forming units at time 0. Results are representative of two assays.

Figure 5

Survival of mice inoculated with the wild-type strain P1/7, M3G, or G6G. Six-week old CD1 mice were intraperitoneally inoculated with 7 × 10⁷ cfu/ml and survival was recorded over a 14-day period. Data are expressed as the mean percentage of live animals in each group (n = 11).