Serotype-specific role of antigen I/II in the initial steps of the pathogenesis of the infection caused by Streptococcus suis

Abstract

Streptococcus suis is one of the most important post-weaning porcine bacterial pathogens worldwide. The serotypes 2 and 9 are often considered the most virulent and prevalent serotypes involved in swine infections, especially in Europe. However, knowledge of the bacterial factors involved in the first steps of the pathogenesis of the infection remains scarce. In several pathogenic streptococci, expression of multimodal adhesion proteins known as antigen I/II (AgI/II) have been linked with persistence in the upper respiratory tract and the oral cavity, as well as with bacterial dissemination. Herein, we report expression of these immunostimulatory factors by S. suis serotype 2 and 9 strains and that AgI/II-encoding genes are carried by integrative and conjugative elements. Using mutagenesis and different in vitro assays, we demonstrate that the contribution of AgI/II to the virulence of the serotype 2 strain used herein appears to be modest. In contrast, data demonstrate that the serotype 9 AgI/II participates in self-aggregation, induces salivary glycoprotein 340-related aggregation, contributes to biofilm formation and increased strain resistance to low pH, as well as in bacterial adhesion to extracellular matrix proteins and epithelial cells. Moreover, the use of a porcine infection model revealed that AgI/II contributes to colonization of the upper respiratory tract of pigs. Taken together, these findings suggest that surface exposed AgI/II likely play a key role in the first steps of the pathogenesis of the S. suis serotype 9 infection.

Figure 1

Characteristics of AgI/II proteins present in different streptococci. The leader peptide signal (LP), N-terminal domain (N-term), alanine-rich region (A), variable region (V), proline-rich region (P), and C-terminal domain, and the cell wall anchorage domain (CWA) containing the LPXTG domain are illustrated for the S. suis AgI/II, S. pyogenes AspA, S. mutans SpaP, and S. gordonii SspA and SspB. Amino acid (aa) size and percentage of S. suis AgI/II protein identity are also indicated. Black arrows indicate the location of primers pET151_S2agI/IIΔCWA_F and pET151_S2agI/II _{_}ΔLPXTG_R, which were used to produce the his-tagged recombinant AgI/II protein, rAgI/II.

Figure 2

The AgI/II protein is expressed in the S. suis serotype 2 and 9 wild-type strains but is absent in S2Δ agI/II and S9Δ agI/II mutant strains. Western blot using cell wall extracts from S. suis serotype 2 (wells 1–3) and serotype 9 (wells 4–6): serotype 2 wild-type strain SC84 (well 1) and serotype 9 wild-type strain 1135776 (well 4); mutant strains S2ΔagI/II (well 2) and S9ΔagI/II (well 5); and complemented strains S2CΔagI/II (well 3) and S9CΔagI/II (well 6). Expected bands at approximately 180 kDa, shown by the black arrow, were observed for the serotype 2 and 9 wild-type and complemented strains, similar to that obtained with the purified AgI/II protein, rAgI/II (well 7), used as a positive control. MW: molecular weight marker.

Figure 3

The S. suis serotype 9 (S9) AgI/II, but not that of the serotype 2 (S2), is implicated in bacterial self-aggregation and biofilm formation. The role of the S. suis AgI/II was evaluated with regards to cell-to-cell aggregation in fluid phase (A) and biofilm formation capacity in the presence of porcine fibrinogen (B) after 24 h of incubation at 37 °C. Data represent the mean ± SEM from at least three independent experiments. **(p < 0.01) and ***(p < 0.001) indicate a significant difference between the S. suis S9 wild-type or complemented strain (S9CΔagI/II) and agI/II-deficient mutant (S9ΔagI/II).

Figure 4

Porcine salivary agglutinins (pSAGs) aggregate S. suis serotype 2 (S2) and serotype 9 (S9). Evaluation of the fluid phase aggregation of the wild-type S. suis S2 and S9 strains in the absence (−) or presence (+) of pSAGs. Aggregation in the absence of pSAGs reflects self-aggregation only. Data represent the mean ± SEM from at least three independent experiments. *(p < 0.05) and **(p < 0.01) indicate a significant difference of S. suis S2 or S9 aggregation in the absence and presence of pSAGs.

Figure 5

The S. suis serotype 2 (S2) and serotype 9 (S9) AgI/II are involved in adhesion to fluid phase porcine salivary agglutinins (pSAGs), but only for S9 with surface-immobilized pSAGs. Evaluation of the fluid phase aggregation of S2 (A) and S9 (B) strains to pSAGs or to surface-immobilized gp340-derived peptide SRCRP2 by S2 (C) and S9 (D), the latter being measured by ELISA. Data represent the mean ± SEM from at least three independent experiments. *(p < 0.05) and **(p < 0.01) indicate a significant difference between the S. suis S2 or S9 wild-type or complemented strain (S2CΔagI/II or S9CΔagI/II) and the agI/II-deficient mutants (S2ΔagI/II or S9ΔagI/II).

Figure 6

The S. suis serotype 9 (S9) AgI/II, but not that of serotype 2 (S2), is involved in protection against acid stress. Effect of acid stress on S. suis S2 and S9 viability, determined at pH 3 (A, B) and pH 5 (C, D). Data represent the mean ± SEM from at least three independent experiments. *(p < 0.05) indicates a significant difference between the S. suis S9 wild-type or complemented strain (S9CΔagI/II) and agI/II-deficient mutant (S9ΔagI/II).

Figure 7

The S. suis serotype 9 (S9) AgI/II and, to a lesser extent, that of serotype 2 (S2), are bacterial adhesins for extracellular matrix proteins. Adhesion of the S. suis S2 and S9 strains to different concentrations of collagen I (A, B), fibrinogen (C, D), and plasma fibronectin (E, F) as evaluated by ELISA. Data represent the mean ± SEM from at least three independent experiments. *(p < 0.05) and **(p < 0.01) indicate a significant difference between the wild-type or complemented strain (CΔagI/II) and the agI/II-deficient mutant (ΔagI/II).

Figure 8

The S. suis serotype 9 (S9) AgI/II, but not that of the serotype 2 (S2), is involved in adhesion to porcine tracheal epithelial cells. Adhesion of the S. suis S2 and S9 strains to NPTr cells after 2 h of incubation with a multiplicity of infection of 10. Data represent the mean ± SEM from at least three independent experiments. *(p < 0.05) indicates a significant difference between the S. suis S9 wild-type or complemented strain (S9CΔagI/II) and the agI/II-deficient mutant (S9ΔagI/II).

Figure 9

The S. suis serotype 9 (S9) AgI/II is implicated in colonization of the porcine respiratory tract. An intranasal porcine model of infection was used to determine the implication of the S. suis S9 AgI/II in colonization of the nasal cavity (A) and tonsils 21 days post-infection (B). Data represent the mean ± SEM from at least three independent experiments. *(p < 0.05) and **(p < 0.01) indicate a significant difference between presence of the S. suis S9 wild-type strain and the agI/II-deficient mutant (S9ΔagI/II).