Neutrophil Extracellular Traps Evasion Mechanism Of Streptococcus Suis Serotype 2

Swine streptococcosis is a common swine infectious disease.It occurs all over the world and it is an important bacterial infectious disease that has been plaguing the development of the pig industry in China.The main clinical manifestations are acute sepsis,meningitis,and chronic infection such as arthritis and endocarditis.Both Streptococcus suis(S.suis)and Streptococcus equi subsp.zooepidemicus(SEZ)are the main pathogens of swine streptococcosis,andStretococcus suisserotype 2(SS2)is one of the most virulent and the inost frequently isolated serotype worldwide.In recent years,the prevalence of swine streptococcosis has developed fast in China.Particularly,swine streptococcosis can occurs secondary to many diseases such as porcine reproductive and respiratory syndrome(PRRS),Porcine Circovirus Disease and haemophilus parasuis,causing serious economic losses.Significantly,SS2 and SEZ are important zoonotic pathogens,which seriously threaten the public health.Neutrophils are the first defense line of innate immunity and they are firstly recruited to the inflammation site and kill pathogens via phagocytosis and degranulation when host is infected.In recent years,Neutrophil extracellular traps(NETs)have been found to trap pathogens and increase the concentration of antibacterial substances to effectively kill pathogens through reticular fiber-like structures.In this study,we investigate mechanisms of Swine Streptococcosis pathogens evading the innate immune response and leading to bacteremia from two aspects:degradation of NETs and inhibition of the formation of NETs.SS2 transposon mutant library was used to screen SS2 virulence factors which inhibit NETs formation,and explore its molecular mechanism.This study will provide novel ideas to clarify the mechanism of SS2 evading innate immunity and to further explore the pathogenic mechanism of SS2.1.The molecular mechanism of SS2 inducing NETs formationSS2 can induce the formation of NETs in vivo and in vitro and its mechanism is not clear.This study found that SS2 incubated with neutrophils can stimulate the production of reactive oxygen species(ROS)and improve the transcriptional levels of TLR2 and TLR4 and the activation of p38 MAPK and ERK1/2 MAPK.Blocking the generation of ROS and TLR4 signaling can inhibit SS2 induced phosphorylation of p38 and ERK1/2 and the formation of NETs.Pretreated neutrophils with p38 MAPK and ERK inhibitors decrease SS2-induced NETs formation significantly.This study confirms that SS2 is recognized by TLR2 and/or TLR4 in host,producing ROS,and then activates neutrophil p38 MAPK and ERK1/2 to induce NETs formation.2.Identification and mechanism of SS2 virulence factors on NETs inhibitionIn this study,six mutant strains with enhanced ability to induce NETs formation in SS2 transposon insertion mutant library were identified via NETs quantitative and immunofluorescence staining techniques.Tn524 is identified and explored in the following study.Gene was inactivated by the insertion of transposon TnYLB-1 and the inactivated gene of Tn524 was identified as SS2 cell surface protein(CSP)by indirect PCR amplification and sequencing.The recombinant protein CSP could not induce the formation of NETs and could not inhibit PMA inducing NETs formation.The pull-down assay indicated that CSP protein did not bind to cellular proteins directly.The ?CSP mutant had a lack of bacterial capsules and an enhanced ability of inducing the formation of ROS and NETs in neutrophils.Compared with the parent strain SS2,?CSP induced a significant upregulation of TLR2 and TLR4 transcription levels and ERK1/2 phosphorylation in neutrophils.However,there was no difference in p38 phosphorylation between ?CSP and parent SS2 treated neutrophils.?CSP improve the transcriptional levels of cytokines which positively regulate NETs formation.In summary,in the course of SS2 infection,CSP protein cannot stimulate or inhibit the formation of NETs directly.However,CSP can protect SS2 from being recognized by TLR2 and/or TLR4 in neutrophils by promoting the synthesis of SS2 capsule,and decrease the transcriptional level of cytokines,and inhibit the generation of ROS and suppress the activation of ERK1/2 in neutrophils,and ultimately inhibit the formation of NETs.3.SS2 biofilms inhibit the formation of NETsThis study found SS2 is more liable to form biofilms in vitroin the presence of neutrophil infiltration.Biofilm isolated from the liver,spleen and kidney of healthy mice challenged with planktonic SS2 using biofilm selective medium,which indicating that SS2 could form bacterial biofilms during the process of infection.Both planktonic and biofilm SS2 induced neutrophils chemotaxis to the infection site,which provides an environment for the interaction between neutrophils and pathogens.The bacteria in biofilms were not phagocytized,however,SS2 biofilm can be killed by NETs.The bactericidal efficacy of NETs on the biofilms and planktonic SS2was equal.Significantly,SS2 biofilm extracellular matrix can inhibit NETs release.This knowledge advances the understanding of biofilms and may aid in the development of treatments for persistent infections with a biofilm component.4.NETs degradation by extracellular nucleases of SEZExtracellular nucleases of pathogens can degrade the DNA backbone of NETs.These nucleases have two characters:(1)secreted extracellular or anchored on the outer membrane,(2)nuclease activity.SS2 can secret extracellular nucleases to degrade portion of NETs.SEZ is also an important pathogen of swine streptococcosis leasing to acute sepsis.Whether SEZ has the similar functional nucleases or not needs further study.In this study 7 genes were found to have these two characteristics and the transcript levels of the genes SESEC_RS04165(product nuclease,ENuc)and SESEC_RS05720(product 5'-nucleosidase,5Nuc)are significantly higher than the other five genes.Prokaryotic expression of ENuc and 5Nuc recombinant proteins are rENuc and r5Nuc.The two proteins have both nuclease activity and nucleosidase activity,which directly degrade calf thymus DNA and NET DNA backbone to deoxyadenosine.Furthermore,deoxyadenosine impairs phagocytosis but not the intracellular bactericidal activity of macrophages.The ?ENuc?5Nuc double-mutant strain demonstrated lower virulence than wild-type SEZ,?ENuc and ?5Nuc single-mutant strains.?ENuc?5Nuc was not able to traverse the bloodstream and transfer to other organs.The three mutant induced fewer NETs and were difficult to survive in NETs.Unlike the nucleases of SS2,ENuc and 5Nuc,important virulence factors,provide SEZ with the ability to degrade NETs completely.They are benefit for bacteria to evade immune system and perturb immune cells by degrading host components to cytotoxic substrate deoxyadenosine.