Identification of a novel virulence determinant with serum opacification activity in Streptococcus suis

Abstract

Streptococcus suis serotype 2 is a porcine and human pathogen with adhesive and invasive properties. In other streptococci, large surface-associated proteins (>100 kDa) of the MSCRAMM family (microbial surface components recognizing adhesive matrix molecules) are key players in interactions with host tissue. In this study, we identified a novel opacity factor of S. suis (OFS) with structural homology to members of the MSCRAMM family. The N-terminal region of OFS is homologous to the respective regions of fibronectin-binding protein A (FnBA) of Streptococcus dysgalactiae and the serum opacity factor (SOF) of Streptococcus pyogenes. Similar to these two proteins, the N-terminal domain of OFS opacified horse serum. Serum opacification activity was detectable in sodium dodecyl sulfate extracts of wild-type S. suis but not in extracts of isogenic ofs knockout mutants. Heterologous expression of OFS in Lactococcus lactis demonstrated that a high level of expression of OFS is sufficient to provide surface-associated serum opacification activity. Furthermore, serum opacification could be inhibited by an antiserum against recombinant OFS. The C-terminal repetitive sequence elements of OFS differed significantly from the respective repeat regions of FnBA and SOF as well as from the consensus sequence of the fibronectin-binding repeats of MSCRAMMs. Accordingly, fibronectin binding was not detectable in recombinant OFS. To investigate the putative function of OFS in the pathogenesis of invasive S. suis diseases, piglets were experimentally infected with an isogenic mutant strain in which the ofs gene had been knocked out by an in-frame deletion. The mutant was severely attenuated in virulence but not in colonization, demonstrating that OFS represents a novel virulence determinant of S. suis.

FIG. 1.

Sequence analysis of OFS. (A) Schematic representation of putative OFS domains. (B) Alignment of partial sequences of OFS (GenBank accession no. AAX56334), SOF of M type 22 (accession no. AAA85219), and FnBA (accession no. CAA80121) to show the representative high homology of these proteins at their N termini. (C) Alignment of C-terminal repetitive sequences (R₁, R₂, and R_3Δ) of OFS. (D) Alignment of the first repeat of OFS (R₁), the MSCRAMM consensus sequence (18), and the partial SfbI sequence (GenBank accession no. CAA48133).

FIG. 2.

Serum opacification activity of recombinant OFS. Shown are domains and amino acids of OFS included in the recombinant constructs (A) encoded by pQE173 (rOFS), pQE173Δrep (rOFSΔrep), and pQE173rep (rOFSrep); their differentiation in Western blots with polyclonal anti-rOFS (C) and anti-rOFSrep (D) sera; and detection of serum opacification activity in an SDS-PAGE overlay assay (E). A Western blot developed with preimmune serum is shown for comparison (B). Positions of molecular weight standards (in thousands) are indicated in the left margin. Lanes (lysates of E. coli M15 [B to D]): 1, pQE31 (with IPTG); 2, pQE173 (without IPTG); 3, pQE173 (with IPTG); 4, pQE173rep (without IPTG); 5, pQE173rep (with IPTG); 6, pQE173Δrep (without IPTG); 7, pQE173Δrep (with IPTG).

FIG. 3.

Expression of ofs RNA. Northern blot analysis of S. suis strain 10 (lane 1), capsular mutant 10cpsΔEF (lane 2), and ofs mutants 10Δofs::ermK1 (lane 3), 10cpsΔEFΔofs::ermK5 (lane 4), 10Δofs3/5 (lane 5), and 10Δofs2/12 (lane 6) with ofs (A), gdh (B), and cps2F (C) as hybridization probes is shown. The 2.9-kb and 1.6-kb positions are based on 23S and 16S rRNA bands, respectively, in the ethidium bromide-stained gel.

FIG. 4.

(A) OFS-mediated serum opacification activity in ofs⁺ S. suis strains (10 and 10cpsΔEF) and in L. lactis (pOriOFSoptRBS) expressing plasmid-encoded OFS. The 1% SDS extracts of the indicated strains were tested for serum opacification activity with the microwell plate assay as described in Materials and Methods. SOF⁻ (M1) and SOF⁺ (M2 and M49) S. pyogenes strains were included as negative and positive controls, respectively, for serum opacification. The optical density of serum incubated with 1% SDS alone was subtracted from each value (corrected absorbance). Mean values and standard deviations of five independent experiments, each performed in triplicate, are shown. Extracts were either not treated (open bars) or treated with anti-rOFSΔrep antiserum (hatched bars). (B) Plasmid-mediated expression of OFS in L. lactis was detectable in Western blots of SDS extracts with antibodies against rOFSΔrep. Lane 1, L. lactis pOri23; lane 2, L. lactis pOriOFS; lane 3, L. lactis pOriOFSoptRBS.