Isolation, Characterization and Biological Properties of Membrane Vesicles Produced by the Swine Pathogen Streptococcus suis

Abstract

Streptococcus suis, more particularly serotype 2, is a major swine pathogen and an emerging zoonotic agent worldwide that mainly causes meningitis, septicemia, endocarditis, and pneumonia. Although several potential virulence factors produced by S. suis have been identified in the last decade, the pathogenesis of S. suis infections is still not fully understood. In the present study, we showed that S. suis produces membrane vesicles (MVs) that range in diameter from 13 to 130 nm and that appear to be coated by capsular material. A proteomic analysis of the MVs revealed that they contain 46 proteins, 9 of which are considered as proven or suspected virulence factors. Biological assays confirmed that S. suis MVs possess active subtilisin-like protease (SspA) and DNase (SsnA). S. suis MVs degraded neutrophil extracellular traps, a property that may contribute to the ability of the bacterium to escape the host defense response. MVs also activated the nuclear factor-kappa B (NF-κB) signaling pathway in both monocytes and macrophages, inducing the secretion of pro-inflammatory cytokines, which may in turn contribute to increase the permeability of the blood brain barrier. The present study brought evidence that S. suis MVs may play a role as a virulence factor in the pathogenesis of S. suis infections, and given their composition be an excellent candidate for vaccine development.

Fig 1. Transmission electron micrographs of an overnight culture of S. suis P1/7 (panel A) and the membrane vesicle preparation (panel B).

Black arrow: whole bacterium; white arrows: membrane vesicles.

Fig 2. General locations (A) and functions (B) of proteins identified in S. suis P1/7 membrane vesicles by ES MS/MS.

Fig 3. Determination of subtilisin (A) and DNase (B) activities in S. suis membrane vesicles.

*: p < 0.05 compared to negative controls.

Fig 4. Quantification of NET degradation by S. suis membrane vesicles.

NETs were formed by the PMA-stimulated promyelocytic leukemia cell line HL60. *: p < 0.05 compared to the negative control (no NET degradation).

Fig 5. Quantification of NF-κB activation in U937-3xκB-LUC monocytes (A) and macrophage-like cells (B) by S. suis membrane vesicles (0.01 to 40 μg/ml), E. coli LPS (10 μg/ml), and S. suis P1/7 whole bacteria (MOI = 100).

Results were considered significant at §: p < 0.05, *: p < 0.01, and #: p < 0.001 compared to unstimulated cells.

Fig 6. Quantification of CXCL-8 (A), TNF-α (B), and IL-1β (C) secretion by macrophage-like cells stimulated with S. suis membrane vesicles (1 to 40 μg/ml), E. coli LPS (10 μg/ml), and S. suis P1/7 whole bacteria (MOI = 100).

Results were considered significant at *p < 0.05 compared to unstimulated cells.