Contribution of fibronectin-binding protein to pathogenesis of Streptococcus suis serotype 2

Abstract

In the present study we investigated the role of the fibronectin (FN)- and fibrinogen (FGN)-binding protein (FBPS) in the pathogenesis of Streptococcus suis serotype 2 in piglets. The complete gene encoding FBPS from S. suis serotype 2 was cloned in Escherichia coli and sequenced. The occurrence of the gene in various serotypes was analyzed by hybridization studies. The FBPS protein was expressed in E. coli and purified, and binding to human FN and FGN was demonstrated. The induction of antibodies in piglets was studied upon infection. An isogenic mutant unable to produce FBPS was constructed, and the levels of virulence of the wild-type and mutant strains were compared in a competitive infection model in young piglets. Organ cultures showed that FBPS was not required for colonization of the tonsils but that FBPS played a role in the colonization of the specific organs involved in an S. suis infection. Therefore, the FBPS mutant was considered as an attenuated mutant.

FIG. 1.

Schematic presentation of the procedure used to clone the fbps gene of S. suis serotype 2 and the construction of an insertional knockout mutant in S. suis serotype 2. A 5-kb EcoRI fragment was cloned in pGEM7Zf(+), yielding pFBPS7-46. In pFBPS7-47, the 382-bp SalI-SalI fragment of pFBPS7-46 was replaced by a 1.2-kb spectinomycin resistance gene, after the vector was made blunt, to obtain an insertional knockout of fbps.

FIG. 2.

Purity and immunogenicity of FBPS purified under native conditions. SDS-PAGE analysis with SYPRO-orange, a nonspecific protein-staining dye (A), and Western blot analysis with a monoclonal antibody against the six-His tag (B) of 4 μl of E. coli M15 [pQE-30-pREP4-FBPS] lysate (lanes 1) and 165 ng of purified FBPS (lanes 2) were carried out. Convalescent-phase serum raised against S. suis strain 10 was used to test immunogenicity of FPBS present in 4 μl of E. coli M15 [pQE-30-pREP4-FBPS] lysate and 0.5 μg of purified FBPS (C) (lanes 1 and 2). Arrowhead, 64-kDa FPBS; Mw, molecular size marker (in kilodaltons).

FIG. 3.

Binding studies with purified FBPS. (A and B) Gels were probed with FN (A) or FGN (B) at 5 μg/ml. Lanes 1 contain 500 ng of purified FBPS, and lanes 2 contain 500 ng of BSA. (C and D) Lanes 3 and 4 contain 500 ng of purified FBPS. Lanes 3 were probed with FN (C) or FGN (D) at 20 μg/ml, and lanes 4 were only incubated with conjugate without FN or FGN. (E and F) Gels were probed with FN (E) or FGN (F) at 20 μg/ml. Lanes 5 contain 1.8 μg of purified FBPS digested with enterokinase, and lanes 6 contain 500 ng of purified FBPS. The closed arrowhead indicates 64-kDa FBPS; the open arrowhead indicates approximately 55-kDa FBPS without the six-His tag. Mw, molecular size marker (in kilodaltons).

FIG. 4.

Distribution of fbps among various S. suis serotypes. Chromosomal DNA (1 μg) was spotted onto nitrocellulose membrane and hybridized with a ³²P-labeled fbps probe. Serotypes were spotted as indicated. S10, S. suis serotype 2 MRP⁺ EF⁺; T15, S. suis serotype 2 MRP⁻ EF⁻; S17, S. suis serotype 2 MRP⁺ EF* (23).

FIG. 5.

Efficiency of colonization of wild-type and mutant bacteria on various organs of infected pigs. (A) Colonization of the wild-type strain 10 (wt) and the mutant strain 10ΔFBPS of the tonsils. Symbols: ⧫, tonsil from pig 4664; ▪, tonsil from pig 4665; ▴, tonsil from pig 4666; •, tonsil from pig 4668. (B) Colonization of the specific organs. Symbols: ◊ and ⧫, pus from joints from pig 4664; ▴, pus from a joint from pig 4666; •, CNS specimen from pig 4668. Each symbol represents the numbers of wild-type or mutant bacteria isolated from one particular organ, from one piglet.