The Surface-exposed Protein SntA Mediated Immune Evasion Of Streptococcus Suis

Streptococcus suis(S.suis)is recognized as an important swine and human pathogen.Among the 33 serotypes,S.suis serotype 2(SS2)is the most virulent and prevalent one,which also an emerging zoonotic pathogen.Two large-scale outbreaks of severe SS2 infection occurred in 1998 and 2005 in China causing 229 infections,52 deaths in human,and a large of animal infection and deaths.Since 2005,highly infection and mortality rate after S.suis infection in pigs were occurred in our country.In 2005,"Streptococcal toxic shock like syndrome(STSLS)was firstly reported to occur in the human.Therefore,the mechanism causing STSLS becomes a research hotspot in S.suis pathogenesis and important progress has achieved in the past decade.During these processes,the host complement system is an important factor facilitating clearing bacterial pathogens.However,the mechanism of immune evasion of S.suis to survive in blood and spread in organism still remains a lack of systematic research.The complement system acts as a first line of defence against pathogenic intruders and a mediator between the innate and adaptive immune response.Although foreign microorganisms can be rapidly recognized and eliminated by host complement,it also offers many interference sites that can disrupt this balanced network of protein interactions by complement-binding proteins leading to failure of elimination by host.In the present study,plenty of complement binding proteins(CBPs)have been identified from surface proteins and secreted proteins of S.suis.C1 q binding protein SntA is identified to be a heme-containing cell wall-anchored protein,it has nucleotidase and nuclease.SntA is highly conserved in Gram-positive bacteria.The mechanism of SntA-mediated immune escape of SS is studied in this study.1.Identification of complement binding proteins from Streptococcus suis.The complement system consists of more than 50 plasma and cell surface proteins that had the enzyme activity after activation existed in the serum,tissue fluid and cell membrane surface.The complement has three independent but interactive activation pathways: the classical pathway,alternative pathway and lectin pathway.Complement binding proteins(CBPs)in a plenty of pathogen have been identified.These CBPs interact with complement and disturb complement activation cascade through capturing,cleavage and inhibition.Therefore,the identification of complement binding proteins(CBPs)in pathogenic microorganism is very important for complement evasion mechanism research.In the present study,complement C1 q A,C3 a,and C3 b were used as baits to screen the CBPs from the bacterial two-hybrid library of SS2 full ORFome(containing 2100 ORFs of SS2 genome).Two C1 q A binding proteins(B9H01＿RS05865;B9H01＿RS10100);four C3 a binding proteins(B9H01＿RS01440;B9H01＿RS10115;B9H01＿RS10100;B9H01＿RS03990)and one C3 b binding protein(B9H01＿RS09920)were screened.The vital developments could augment our knowledge of interactions of complement and SS.2.The surface-exposed protein SntA contributes to complement evasion in zoonotic Streptococcus suis.In the previous study,the streptococcal heme-binding protein SntA can interact with the host anti-oxidant protein AOP2 and inhibit the host antioxidant activity,leading to pathological damage in infected pigs(Wan et al.,2017).In the present study,SntA can also bind to complement C1 q,suggesting that SntA may mediate SS complement evasion or complement activation.To further study the role of SntA in complement activation or evasion,the complementary strain CΔsnt A was constructed based on the snt A-deletion mutant strain(Δsnt A)in our lab.Mouse pathogenesis and competitive colonization experiments found that the pathogenic ability and blood,brain,lung and kidney colonization in the parental strain SC-19 was significantly highly than the Δsnt A strain.Anti-phagocytosis experiments showed that the ability of Δsnt A strains to survive in whole blood,serum,RAW264.7 phagocytic cells and human PMNs was significantly lower than that of parent strain SC-19 and complementary strain CΔsnt A,revealing that SntA has significant anti-phagocytic ability.Complement deposition and membrane attack complexes formation assays showed that C3 deposition and the formation of membrane attack complexes on the surface of Δsnt A strains were significantly higher than those of SC-19 and CΔsnt A.Complement-mediated hemolysis experiments demonstrated that SntA can significantly inhibit the hemolytic activity mediated by the classical complement activation pathway.Complement activation experiments revealed that SntA protein itself can activate the classical and lectin complement pathway,consequently consuming the complement components.Direct and indirect binding experiments confirmed that SntA can interact with complement C1 q,and inhibite the binding of C1q-Ig G,C1q-antigen-antibody complexes.This study demonstrated that SntA can inhibit the hemolytic activity mediated by the classical complement pathway by binding to C1 q.It can also activate complement in both classical and lectin complement pathway.These two complement evasion mechanisms are crucial for the pathogenesis of S.suis.3.The nucleotidase SntA contributes to the survival of S.suis in blood One of the effective strategies for pathogen to invade host immune system recognition and killing is breaking purine signal pathway.In the present study,snt A was identified to be a homologue of the 5’-nucleotidase of S.suis by bioinformatic analysis.The multiple sequence alignments indicated that SntA had highly amino acid sequence similarity with known 5′-nucleotidase.It also had a characteristic signal sequence of 5′-nucleotidase.These data revealed that SntA was important for S.suis immune evasion.In vitro enzyme activity assays revealed that SntA had ATPase activity and ADPase activity,but did not have AMPase activity.Whole blood and PMNs killing experiments found that5’-nucleotidase inhibitors significantly down-regulated the survival rate of SC-19 and CΔsnt A in whole blood,and no significant effect was observed in Δsnt A(p>0.05);adenosine could significantly up-regulate the survival rate of the Δsnt A strain in whole blood and PMNs.In addition,DNA degradation experiment revealed that SntA also had the ability to hydrolyze double-stranded DNA.This study confirmed that SntA had nucleotidase activity and nuclease activity,and consequently contributed to the survival of S.suis in blood.4.SntA contributes to the environmental adaptability of S.suisIn the pathogenesis of SS2,many proteins directly or indirectly participate in this process,mainly including surface-associated and secreted proteins.Many LPXTG motif-anchored proteins are considered to be virulence factors,but the mechanism of their influence on virulence remains unclear.In the previous study,SntA was identified to be a cell wall-anchored protein with an LPXTG motif and associated with the pathogenesis of S.suis.In the present study,the growth curve assay showed that no significant difference of the growth rate was observed between Δsnt A strain with SC-19 and CΔsnt A on OD and CFU.Stress response experiments revealed that SntA did not affect the ability of S.suis to resist hypertonicity,hypotonicity and high temperature stress.Biofilm formation experiments revealed that SntA did not affect the biofilm formation of S.suis.Transmission electron microscopy revealed no significant changes in the decidua between the parent strains SC-19 and Δsnt A.Scanning electron microscopy showed that the morphology of the Δsnt A strain was significantly changed.Bacterial adhesion and invasion experiments showed that the ability of Δsnt A strains to adhere to and invade Hep-2 epithelial cells was significantly stronger than SC-19 and CΔsnt A.This study demonstrated that SntA affected the morphology of SS and the ability to adhere to and invade host cells.These results further elucidate the effect of SntA on the pathogenesis of S.suis.