Role of Maturation of Lipoproteins in the Pathogenesis of the Infection Caused by Streptococcus suis Serotype 2

Abstract

Streptococcus suis serotype 2 is an important porcine bacterial pathogen associated with multiple pathologies in piglets. Bacterial lipoproteins (LPPs) have been described as playing important roles in the pathogenesis of the infection of other Gram-positive bacteria as adhesins, pro-inflammatory cell activators and/or virulence factors. In the current study, we aimed to evaluate the role of the prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein signal peptidase (Lsp) enzymes, which are responsible for LPP maturation, on the pathogenesis of the infection caused by two different sequence types (STs) of S. suis serotype 2 strains (virulent ST1 and highly virulent ST7). Through the use of isogenic Δlgt, Δlsp and double Δlgt/Δlsp mutants, it was shown that lack of these enzymes did not influence S. suis adhesion/invasion to porcine respiratory epithelial cells. However, in the absence of the Lsp and/or Lgt, a significant reduction in the capacity of S. suis to activate phagocytic cells and induce pro-inflammatory mediators (in vitro and in vivo) was observed. In general, results obtained with the double mutant did not differ in comparison to single mutants, indicating lack of an additive effect. Finally, our data suggest that these enzymes play a differential role in virulence, depending on the genetic background of the strain and being more important for the highly virulent ST7 strain.

Keywords: Streptococcus suis; inflammation; lipoproteins; maturation.

Figure 1

Growth of the S. suis serotype 2 wild-type virulent strain P1/7 (ST1) and highly virulent strain SC84 (ST7) (black) in THB (A,C) and plasma (B,D) as well as their respective Δlgt (blue), Δlsp (red) and Δlgt/Δlsp (purple) mutants. Each point represents mean bacterial concentration (CFU/mL) +/− SEM (n = 3 independent experiments).

Figure 2

Adhesion (A–D) and invasion (E–H) of the S. suis serotype 2 wild-type virulent strain P1/7 (ST1) (A,B,E,F) and highly virulent strain SC84 (ST7) (C, D, G and H) (black) as well as their respective Δlgt (blue), Δlsp (red) and Δlgt/Δlsp (purple) mutant strains to porcine tracheal epithelial cells after 2 h (A,C,E,G) or 4 h (B,D,F,H) of incubation. * (p < 0.05) indicates a significant difference between the ΔcpsF mutant and wild-type strain or the Δlsp and/or Δlgt/Δlsp mutant strains. Each bar represents the mean bacterial concentration (CFU/mL) + SEM (n = 3 independent experiments).

Figure 3

Capacity of the S. suis serotype 2 virulent wild-type ST1 strain P1/7 (A) and highly virulent ST7 strain SC84 (B) (black), and their respective Δlgt (blue), Δlsp (red), Δlsp + pMX1-lsp (dark red) and Δlgt/Δlsp (purple) mutant strains to resist the bactericidal effect of murine whole blood after 2 h of incubation. Percentage of bacterial survival was calculated in comparison to bacteria in plasma alone. Data represent the mean + SEM (n = 3 independent experiments). * (p < 0.05) indicates a significant difference between the wild-type and mutant strains.

Figure 4

Pro-inflammatory mediator production by bmDCs following infection with live bacteria of the S. suis serotype 2 virulent wild-type ST1 strain P1/7 (A–D) and highly virulent ST7 strain SC84 (E–H) (black), as well as their respective Δlgt (blue), Δlsp (red) and Δlgt/Δlsp (purple) mutant strains and the Δlgt + pMX1-lgt complemented strain (dark blue). Production of TNF (A,E), IL-6 (B and F), CXCL1 (C,G) and CCL3 (D,H). Data represent the mean + SEM (n = 4 independent experiments). * (p < 0.05) indicates a significant difference between the wild-type and mutant strains. Mock-infected cells induced negligible cytokine values < 300 pg/mL (not shown).

Figure 5

Pro-inflammatory mediator production by bmDCs following infection with heat-killed bacteria of S. suis serotype 2 virulent wild-type ST1 strain P1/7 ST1 (A–D) and highly virulent ST7 strain SC84 (E–H) (black), as well as their respective Δlgt (blue), Δlsp (red) and Δlgt/Δlsp (purple) mutant strains and the Δlgt + pMX1-lgt (dark blue) or Δlsp + pMX1-lgt complemented strains (dark red). Production of TNF (A,E), IL-6 (B and F), CXCL1 (C,G) and CCL3 (D and H). Data represent the mean + SEM (n = 4 independent experiments). * (p < 0.05) indicates a significant difference between the wild-type and mutant strains. Mock-infected cells induced negligible cytokine values < 300 pg/mL (not shown).

Figure 6

Pro-inflammatory mediator production by bmDCs following infection with bacterial-free supernatant of S. suis serotype 2 virulent wild-type ST1 strain P1/7 (A–D) and highly virulent ST7 strain SC84 (E–H) (black), as well as their respective Δlgt (blue), Δlsp (red) and Δlgt/Δlsp (purple) mutant strains and the Δlgt + pMX1-lgt (dark blue) or Δlsp + pMX1-lgt complemented strains (dark red). Production of TNF (A,E), IL-6 (B,F), CXCL1 (C,G) and CCL3 (D,H). Data represent the mean + SEM (n = 4 independent experiments). * (p < 0.05) indicates a significant difference between wild-type and mutant strains. Mock-infected cells induced negligible cytokine values < 300 pg/mL (not shown).

Figure 7

Survival (A,D) and blood bacterial burden at 12 h (B,E) and 24 h post-infection (C,F) of C57BL/6 mice following intraperitoneal inoculation of the S. suis serotype 2 virulent wild-type ST1 strain P1/7 and highly virulent ST7 strain SC84 (black) as well as their respective Δlgt (blue), Δlsp (red) and Δlgt/Δlsp (purple) mutant strains. Data represent survival curves (A,D) (n = 15) or geometric mean (B,C,E,F) (n = survived mice at each time point). * (p < 0.05) indicates a significant difference between survival or blood bacterial burden of mice infected mutant strains when compared to their respective wild-type strain.

Figure 8

Plasma levels of IL-6 (A), IL-12p70 (B), G-CSF (C), IFN-γ (D), CCL2 (E), CCL3 (F), CCL4 (G), CCL5 (H), CXCL9 (I) and CXCL2 (J) in mice 12 h following intraperitoneal inoculation of the S. suis serotype 2 virulent wild-type ST1 strain P1/7 (black) or Δlgt (blue), Δlsp (red) and Δlgt/Δlsp (purple) mutant strains. Data represent mean + SEM (n = 8 individuals). * p < 0.05 indicates a significant difference between plasma levels of mice infected with the mutant strains when compared to the wild-type strain.

Figure 9

Plasma levels of IL-6 (A), IL-12p70 (B), G-CSF (C), IFN-γ (D), CCL2 (E), CCL3 (F), CCL4 (G), CCL5 (H), CXCL9 (I) and CXCL2 (J) in mice 12 h following intraperitoneal inoculation of the S. suis serotype 2 highly virulent wild-type ST7 strain SC84 (black) or Δlgt (blue), Δlsp (red) and Δlgt/Δlsp (purple) mutant strains. Data represent mean + SEM (n = 8 individuals). * p < 0.05 indicates a significant difference between plasma levels of mice infected with the mutant strains when compared to the wild-type strain.