Primary Study On The Mechanism Of Streptococcus Suis Serotype 2 Inducing The Formation Of Neutrophil Extracellular Traps

Streptococcus suis(S. suis) is an important zoonotic pathogen. S. suis infected pigs mainly show arthritis, meningitis, pneumonia, sepsis and other clinical symptoms. The two outbreaks of Streptococcus suis type 2(SS2) in Jiangsu(1998) and Sichuan(2005) province emerged human infection cases causing serious clinical symptoms, and even leading to death. This further indicates that SS2 is an important zoonotic pathogen which leads to serious public health threaten.Neutrophils are the primary activated immune cells in bacterial infections and play very important role in controlling of SS2 infection. The antibacterial patterns of neutrophils include phagocytosis, degranulation and neutrophil extracellular traps(NETs). When stimulated by specific substances or pathogens, neutrophils release their entire nucleus into the surrounding tissues and decorate bactericidal proteins on the DNA fibers. This structure of DNA fibers decorated with bactericidal proteins is denoted as neutrophil extracellular traps. The traps can efficiently capture pathogens and further kill them. This anti-microbial mechanism has been widely accepted.Previous studies showed that high virulent strains of SS2 can effectively resist killing by neutrophil phagocytosis and degranulation. However, the bactericidal effect of NETs during SS2 infection has rarely reported. Therefore, studying on the mechanism of the NETs bacterial effect on SS2 will make great contribution to revealing the pathogenic mechanism of SS2. The present study describes the bacterial effect of NETs on SS2 infection in vitro and in vivo. And we further explored the mechanism of SS2 inducing the formation of NETs by label-free quantitative proteomics.Firstly, in order to detect the role of NETs plays in SS2 strain 05 ZY infection, mice were infected with SS2 strain 05 ZY. The results showed that SS2 infected mice can form neutrophil extracellular traps and the treatment of nuclease destroyed this structure. Therefore, this study used nuclease evaluates the role of NETs in 05 ZY infection. The results showed that nuclease treatment destroyed NETs structure. Mice treated with nuclease showed significant higher bacteria load in blood, higher proinflammatory cytokines TNF-αlevel, much severe clinical symptoms and much higher mortality. This suggested that NETs played important role in bacteria clearance and inflammation controlling and further contribute the survival of mice infected with 05 ZY.Although NETs had protective effect on mice during 05 ZY infection, there were still considerable part of mice dying of 05 ZY infection, this showed that there may be a mechanism for SS2 to escape NETs killing. In order to explain this phenomenon, this study used in vitro experiment to detect the mechanism of NETs escaping from SS2. This study found that the different virulent SS2 strains can induce the formation of NETs in vitro. And SS2 strains can evade phagocytosis by murine neutrophils. However, the capsular polysaccharide(CPS) knock-out mutant of SS2 can effectively be swallowed. Further comparison of the killing efficacy of SS2 strains by NETs showed that, SS2 strains can effectively evade NETs killing, but the CPS knock-out mutant can be efficiently killed by NETs. Accordingly, the efficacy of NETs trapping on CPS knock-out mutant was much higher than that of SS2 strains. This suggested that the CPS structure contributed SS2 strains to evading the killing not only by phagocytosis but also by neutrophil extracellular traps.Because the mechanism of NETs formation is still unclear, we further analysis the mechanism of NETs formation induced by SS2. This study detected the protein expression of normal neutrophils, PMA stimulated neutrophils and SS2 strain 05 ZY infected neutrophils by label-free quantitative proteomics. The proteomics results showed there were 75% detected proteins showed similar differential expression in the two stimulated groups. These proteins mainly involved in nucleotide binding, RNA binding, ATP binding and other life process. The MPO activity, ROS production, PKC pathways are involved in the formation of NETs stimulated by PMA and 05 ZY. Biological process analysis showed that there was a large number of proteins involved in the reorganization of the actin cytoskeleton. This study used cytochalasin B, the inhibitor of actin polymerization to study the role of actin in NETs formation. The results showed that PMA induced NETs levels were significantly decreased after treatment with cytochalasin B, but no significant effect on NETs levels induced by SS2. The comparison of protein amount involved in molecular function of PMA stimulated and 05 ZY infected groups showed that the number of proteins involved in endopeptidase activity in PMA stimulated group was significantly higher than that in the 05 ZY stimulated group. One of these proteins is matrix metalloproteinase MMP8 which co-located with the NETs structure, but its specific role in NETs formation and mechanism need to be further studied.