Study of the Role of Lipoprotein Maturation Enzymes in the Pathogenesis of the Infection Caused by the Streptococcus suis Serotype 2 Sequence Type 25 North American Prototype Strain

Abstract

Streptococcus suis serotype 2 is an important swine bacterial pathogen causing sudden death, septic shock, and meningitis. However, serotype 2 strains are phenotypically and genotypically heterogeneous and composed of a multitude of sequence types (STs) whose distributions greatly vary worldwide. It has been previously shown that the lipoprotein (LPP) maturation enzymes diacylglyceryl transferase (Lgt) and signal peptidase (Lsp) significantly modulate the inflammatory host response and play a differential role in virulence depending on the genetic background of the strain. Differently from Eurasian ST1/ST7 strains, the capsular polysaccharide of a North American S. suis serotype 2 ST25 representative strain only partially masks sub-capsular domains and bacterial wall components. Thus, our hypothesis is that since LPPs would be more surface exposed in ST25 strains than in their ST1 or ST7 counterparts, the maturation enzymes would play a more important role in the pathogenesis of the infection caused by the North American strain. Using isogenic Δlgt and Δlsp mutants derived from the wild-type ST25 strain, our studies suggest that these enzymes do not seem to play a role in the interaction between S. suis and epithelial and endothelial cells, regardless of the genetics background of the strain used. However, a role in the formation of biofilms (also independently of the STs) has been demonstrated. Moreover, the involvement of LPP dendritic cell activation in vitro seems to be somehow more pronounced with the ST25 strain. Finally, the Lgt enzyme seems to play a more important role in the virulence of the ST25 strain. Although some differences between STs could be observed, our original hypothesis that LPPs would be significantly more important in ST25 strains due to a better bacterial surface exposition could not be confirmed.

Keywords: North America; Streptococcus suis; inflammation; lipoprotein maturation enzymes; serotype 2.

Figure 1

Bacterial hydrophobicity is not affected by the absence of Lgt or Lsp and both defective mutants grow as well as the wild-type strain in plasma. Surface hydrophobicity using n-hexadecane (A) and bacterial growth in THB (B) and plasma (C) of S. suis serotype 2 wild-type 89-1591 (ST25) strain, Δlgt (blue), and Δlsp (red) mutants (n = 3 independent repetitions for each experiment). The non-encapsulated 89-1591 ΔcpsF was used as a positive control for the hydrophobicity test. * (p < 0.05) indicates a significant difference between wild-type and ΔcpsF mutant strain.

Figure 2

The lack of lipoprotein maturation enzymes does not change the capacity of adhesion and invasion of S. suis to swine epithelial and endothelial cells. Adhesion (A) and invasion (B) of the S. suis 2 wild-type strain 89-1591 (ST25) as well as its respective Δlgt (blue) and Δlsp (red) mutant strains to swine tracheal epithelial cells. Adhesion and invasion of the S. suis 2 wild-type P1/7 (ST1) strain (C,D) and 89-1591 (ST25) strain (E,F) as well as their respective Δlgt (blue) and Δlsp (red) mutant strains to brain microvascular endothelial cells. * Indicates a significant difference (p < 0.05). Each bar represents the mean bacterial concentration (CFU/mL) + SEM from at least three independent experiments.

Figure 3

The Lgt and Lsp enzymes are both implicated in biofilm formation by S. suis independently of the ST of the strain. Biofilm formation capacity in the presence of porcine fibrinogen after 24 h of incubation at 37 °C of S. suis serotype 2 wild-type strain P1/7 (ST1) (A) and strain 89-1591 (ST25) (B) as well as their respective Δlgt (blue), Δlsp (red) mutant, the 89-1591 comp Δlgt (dark blue), and 89-1591 comp Δlsp complemented strains (dark red). Data represent the mean ± SEM from at least three independent experiments. * Indicates a significant difference with the respective wild-type strain (p < 0.05).

Figure 4

The diacyl motif is important for recognition by innate immune cells of periplasmic and/or secreted S. suis serotype 2 ST25 lipoproteins. Pro-inflammatory mediator production by bmDCs following activation with live bacteria of the S. suis serotype 2 wild-type strain 89-1591 (ST25) (A–D) (black), as well as their respective Δlgt (blue) and Δlsp (red)) mutant strains and the Δlgt + pMX1-lgt complemented strain (dark blue). Production of TNF (A), IL-6 (B), CXCL1 (C), and CCL3 (D). 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.

Figure 5

The diacyl motif is important for recognition by innate immune cells of periplasmic S. suis serotype 2 ST25 lipoproteins. Pro-inflammatory mediator production by bmDCs following infection with heat-killed bacteria of the S. suis serotype 2 wild-type ST25 strain 89-1591 (ST25) (A–D) (black), as well as its respective Δlgt (blue) and Δlsp (red)) mutant strains and the 89-1591 comp Δlgt complemented strain (dark blue). Production of TNF (A), IL-6 (B), CXCL1 (C), and CCL3 (D). 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.

Figure 6

Both the diacyl motif and the peptide signal cleavage are important for recognition by innate immune cells of secreted S. suis serotype 2 ST25 lipoproteins. Pro-inflammatory mediator production by bmDCs following infection with bacterial-free supernatant of S. suis serotype wild-type ST25 strain 89-1591 (black) as well as its respective Δlgt (blue) and Δlsp (red) mutant strains and the 89-1591 comp Δlgt (dark blue) or 89-1591 comp Δlsp complemented strains (dark red). Production of TNF (A), IL-6 (B), CXCL1 (C), and CCL3 (D). 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.

Figure 7

Presence of Lgt, but not Lsp, is significantly important for S. suis ST25 virulence but it does not affect bacteremia levels. Survival (A) and blood bacterial burden at 12, 24 h and 48 h post-infection (B–D) of C57BL/6 mice following intraperitoneal inoculation of the S. suis wild-type 89-1591 strain (ST25) (black), and Δlgt (bleu) and Δlsp (red) mutant strains. Data represent survival curves (A) (n = 15) or geometric mean (B–D) (n = survived mice at each time point). * p < 0.05 indicates a significant difference between survival of mice infected the wild-type and the Δlgt mutant strain.

Figure 8

Lgt and Lsp enzymes are important for the recognition of lipoproteins and therefore for the establishment of the inflammatory response. Plasma levels of IL-6 (A), G-CSF (B), CCL2 (C), CCL3 (D), CCL4 (E), CCL5 (F), CXCL9 (G), and CXCL2 (H) in mice 12 h following intraperitoneal inoculation of S. suis serotype 2 wild-type ST25 strain 89-1591 (black) or its Δlgt (blue) and Δlsp (red) 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 those infected with the wild-type strain.