Superoxide Dismutase SodA And A Serine Threonine/phosphatase Stp1 Of Streptococcus Suis Type 2 Are Critical For Its Virulence

Streptococcus suis type 2 (SS2) is a zoonotic agent of pigs and human, characterized as meningitis, arthritis, septicemia and even toxic syndrome leading to death. There were two major outbreaks of SS2 infections in human in Jiangshu and Sichuan provinces in 1998 and 2005 due to direct contact with infected pigs or carcasses. SS2 is emerging as a threat to public health. The pathogenesis of SS2 infection is closely related to its adhesive and invasive capability to host respiratory epithelial cells and vascular endothelial cells, resistance to macrophage phagocytosis and activation of immune cells in the inflammatory response. The surface proteins of SS2 can bind to regulatory factors in human serum for the bacterium to escape phagocytic clearance. Bacterial components such as capsular polysaccharide, peptidoglycan deacetylase protein (pgdA), superoxide dismutase A (SodA), contribute to resistance to macrophage phagocytosis. SodA is an important virulence factor of SS2 by protecting the bacterial cells from destruction by reactive oxygen species (ROS). Recent studies have revealed that prokaryotic serine/threonine kinases and phosphatases play important roles in resistance to environmental stresses and regulation of bacterial virulence factors involved in pathogenicity.This study attempted to (1) investigate the mechanisms of increased adhesion of SS2 to host cells due to sodA deletion; (2) explore the mechanisms of lessened autophagic response in cells infected with sodA deletion mutant; (3) examine the role of the putative serine/threonine phosphatase Stpl in virulence to mice.1. Streptococcus suis type 2 utilizes SodA for enhanced adhesion to host cells and lessened autophagic responseA sodA deletion mutant (△sodA) was constructed by homologous recombination. Adhesion of the AsodA mutant was significantly higher than the wild-type strain (WT) in bEnd.3 (22.6‰ vs 0.3‰,p＜0.01) and in HEp-2 (21.3%o vs 0.4%o, p<0.01). The AsodA mutant was more easily phagocyted than the WT (15.71% vs 0.67%, p<0.01) in RAW264.7 macrophage cells. To explore the mechanisms of the increased adhesion of the AsodA mutant. We compared the surface proteins of WT and AsodA strains by two-dimensional electrophoresis. Six proteins were analysed by mass spectrography and their transcription levels measured by quantitative real-time PCR. Results showed that the AsodA mutant had a greatly increased mRNA transcription level of thioredoxin-1 and thioredoxin-2, as compared to WT. Using rabbit polyclonal antibodies to probe the expression level by Western blotting, only thioredoxin-2 was increased in the AsodA mutant than in WT, suggesting that thioredoxin-2 might contribute to increased cell adhesion.Data have recently shown that superoxide dismutase (SodA) of SS2 is a virulence factor acting by increasing resistance of the bacterial cells to oxidative stresses. Reactive oxygen species, products of the respiratory burst of phagocytic cells, have been shown to activate autophagy. In order to understand if and how SS2 explores its SodA to interfere with cell autophagic responses, a sodA deletion mutant (△sodA) was compared with its parent and complemented strain in autophagic response in the murine macrophage cell line RAW264.7 (MOI 100:1). The AsodA mutant induced significant autophagic responses in infected cells, as shown by increased LC3 lipidation (LC3-Ⅱ) and EGFP-LC3 punctae, as compared to those infected by the parent or complemented strain. Co-localization of the autophagosomal EGFP-LC3 vesicles with lysosomes was seen in the cells infected with AsodA mutant and its parent strain. Enhaced autophagic responses of the cells infected with AsodA mutant might be related to increased ROS within the cells due to deletion of SodA, as measured by flow cytometric methods using the probes Dihydroethidium,DHE and dihydrodichlorofluorescein diacetate,H2DCF-DA. There was no difference of H2O2 levels between the two strains. We also found that deletion of sodA led to decreased resistance to oxidative stress. SodA expressed in Escherichia coli had SOD activity and could clear ROS in the cell free xanthine oxidase-hypoxanthine ROS-generating system. ROS and inflammatory responses are known to regulate autophagy. Therefore, we propose that SS2 makes use of its sodA for survival not only by scavenging ROS but also by alleviating the host autophagic responses due to ROS stimulation.2. Streptococcus suis type 2 Stpl functions as a phosphatase and involved in virulence to miceSS2 contains a putative serine/threonine protein phosphatases 1 (Stpl) encoded by stpl. Such phosphatases are metalloenzymes and dephosphorylate their substrates in a single reaction step using a metal-activated nucleophilic water molecule. To investigate the role of stpl in pathogenesis of SS2, an isogenic stpl mutant (Astpl). The Astpl mutant exhibited significant increase of adhesion to HEp-2 and bEnd.3 cells (1.9‰ vs 0.3%o in bEnd.3, p<0.01; and 1.9‰ vs 0.4%o in HEp-2, p<0.05). Survival within RAW264.7 cells was 0.79% for the Astpl mutant but 0.16% for WT (p<0.05). Increased survival in macrophage cells might be related to resistance to ROS since the Astpl mutant was more resistant to paraquat-induced oxidative stress than its parent strain (p<0.01). However, deletion of stpl significantly attenuated virulence of SS2 to mice, as shown about 1.8-fold increase of the LD50 value in the mouse model. The Astpl mutant was far more easily cleared from the organs than WT. Two dimensional electrophoresis plus Western blotting with anti-serine phosphorylation antibody and mass spectrometry were used to examine phosphoproteomic difference. Data suggested that Stpl might regulate at least 10 proteins related to phosphorylation and dephosphorylation. In vitro phosphorylation assay showed that Stpl expressed in E. coli was able to dephosphorylate pNPP. However, dephosphorylation of protein kinase activated SodA was not yet clearly demonstrated possibly because of methodological issues.In summary, results demonstrate that SS2 makes use of SodA for its adhesion and survival. SodA alleviates the host autophagic response by scavenging ROS. Stp1 plays a role in virulence of SS2 in mouse model. These findings could help better understanding of the pathogenesis of Streptococcus suis type 2.