Capsular polysaccharide switching in Streptococcus suis modulates host cell interactions and virulence

Abstract

The capsular polysaccharide (CPS) of Streptococcus suis defines various serotypes based on its composition and structure. Though serotype switching has been suggested to occur between S. suis strains, its impact on pathogenicity and virulence remains unknown. Herein, we experimentally generated S. suis serotype-switched mutants from a serotype 2 strain that express the serotype 3, 4, 7, 8, 9, or 14 CPS. The effects of serotype switching were then investigated with regards to classical properties conferred by presence of the serotype 2 CPS, including adhesion to/invasion of epithelial cells, resistance to phagocytosis by macrophages, killing by whole blood, dendritic cell-derived pro-inflammatory mediator production and virulence using mouse and porcine infection models. Results demonstrated that these properties on host cell interactions were differentially modulated depending on the switched serotypes, although some different mutations other than loci of CPS-related genes were found in each the serotype-switched mutant. Among the serotype-switched mutants, the mutant expressing the serotype 8 CPS was hyper-virulent, whereas mutants expressing the serotype 3 or 4 CPSs had reduced virulence. By contrast, switching to serotype 7, 9, or 14 CPSs had little to no effect. These findings suggest that serotype switching can drastically alter S. suis virulence and host cell interactions.

Figure 1

Effect of serotype switching on S. suis CPS expression. (a) Hydrophobicity of the different S. suis strains/mutants. Very low surface hydrophobicity is indicative of high encapsulation, which is demonstrated in the previous study. Data are expressed as mean ± standard error of the mean (SEM) (n = 3). An asterisk denotes a significant difference with SS2 by Mann–Whitney rank sum test (p < 0.05). (b) Transmission electron micrographs showing CPS expression of the different S. suis strains/mutants. Scale bars = 0.5 µm.

Figure 2

Mutations present in the generated S. suis serotype-switched mutants. Each of the schematic representations illustrates the analysis data using Geneious Prime mapping of the draft genome sequence of each mutant (upper part) on the publicly available completed genome sequence of serotype 2 (accession no. AM946016) and the sequence alignment between two genomes (lower part). All gaps between the contigs of each mutant were due to multi-copy genes, such as rRNA genes, tRNA genes and IS elements, or repeated regions within genes. Gaps of the repeated regions within genes were found in the genes corresponding to the SS2 locus tags SSU0496, SSU1127, SSU1171, and SSU1172. Detailed data on mutated genes can be found in Supplementary Table S2. Below the bottom panel are displayed the descriptions for each color of the different drawings.

Figure 3

Impact of serotype switching on S. suis adhesion to and invasion of porcine tracheal epithelial cells, resistance to phagocytosis by macrophages, whole blood bacterial killing, and pro-inflammatory mediator production by dendritic cells. Adhesion (a) and invasion (b) of the different S. suis strains and mutants to NPTr porcine tracheal epithelial cells after 2 h of incubation. (c) Internalization of the different S. suis strains and mutants by J774A.1 murine macrophages after 2 h of incubation. (d) Killing of the different S. suis strains and mutants by murine whole blood after 4 h of incubation. (e) Growth capacity of the different S. suis strains and mutants in porcine whole blood after 4 h of incubation. (f) Pro-inflammatory mediator production by DCs at 16 h following infection with the different S. suis strains and mutants as measured by ELISA. Production of tumor necrosis factor (TNF), interleukin (IL)-6, IL-12p70, C-C motif chemokine ligand (CCL) 5, and C-X-C motif chemokine ligand (CXCL) 1, and CXCL9. C-denotes cells in medium alone. All the data represent the mean ± SEM (n = 4). An asterisk denotes a significant difference with SS2 by Mann–Whitney rank sum test (e) (p < 0.05).

Figure 4

Impact of serotype switching on S. suis virulence and plasma pro-inflammatory mediator production in a mouse model of infection. (a) Survival of C57BL/6 mice following intraperitoneal inoculation of 1 × 10⁷ CFU of the different S. suis strains and mutants. (b) Blood bacterial burden 24 h post-infection of C57BL/6 mice. A blood bacterial burden of 2 × 10⁹ CFU/mL, corresponding to average burden upon euthanasia, was attributed to euthanized mice. (c) Plasma levels of IL-6, IL-12p70, IFN-γ, CCL2, CCL3, CCL4, CCL5, and CXCL2 in C57BL/6 mice at 12 h following intraperitoneal inoculation of 1 × 10⁷ CFU of the different S. suis strains and mutants. Data represent survival curves (a) (n = 10–12), geometric mean (b) (n = 10–12) or mean ± SEM (C) (n = 8). An asterisk denotes a significant difference with SS2 by Log-rank (Mantel-Cox) test (c) and Mann–Whitney rank sum test (b and c) (p < 0.05).