Research On Host Immune Evasion Mechanism Of Two-component Signal Transduction System VraSR In Streptococcus Suis Serotype 2

Streptococcus suis is a major porcine pathogen that is mostly associated with serious diseases including septicemia,arthritis,endocarditis,pneumonia and meningitis and sudden death,which lead to severe economic losses worldwide.Among the known 33 serotypes,Streptococcus suis serotype 2（S.suis 2）is the most virulent and prevalent pathogen in the pig industry.At same time,it can also be communicated to human beings through direct contact with infected pigs or contaminated pig byproducts,resulting in meningitis and streptococcal toxic shock-like syndrome（STSLS）in humans.Two-component signal transduction system（TCS）is widely found in bacteria,which regulates various important physiological and metabolic processes and plays an important role in the virulence and pathogenicity of bacteria.However,the TCS regulatory mechanisms used by S.suis 2to defend against the innate immune destruction have not been directly investigated.Together with the two-component gene deletion mutantsΔTCS2（ΔbceRS）、ΔTCS3（ΔnisKR）、ΔTCS4（ΔsalKR）、ΔTCS5（ΔciaRH）、ΔTCS6（ΔvicK）、ΔTCS8（Δihk/rr）、ΔTCS11（Δ1910KR）in our laboratory,the other two-component gene deletion mutantsΔTCS1（ΔvraSR）、ΔTCS7（ΔSC84₁438hk/1439rr）、ΔTCS9（ΔSC84₁523hk/1522rr）、ΔTCS10（ΔcovR）、ΔTCS12（ΔSC84₁840rr）、ΔTCS13（ΔrevS）、ΔTCS14（ΔSC84₁925rr）、ΔTCS15（ΔvirSR）were constructed through homologous recombination.This study selected an essential regulatory system VraSR through the comparison of survival ability of 15 TCS gene deletion mutants in whole human blood and response regulator genes expression after PMN stimulus.This study aims to investigate the role of VraSR in innate immune evasion and virulence in virulence.The main results are as follows:1.Comparison of the survival ability of 15 TCS gene deletion mutants in human bloodIncubation of SC19 and 15 mutants in human blood for 1,2 and 3 h,collected the samples for counting the number of survival bacteria and calculated the survival rate respectively.It showed that wild strain and mutants all continually proliferated in human blood.Compared with WT SC19,which generally maintains a high growth rate in human blood,the growth rate ofΔvraSR andΔihk/rr was significantly slower.2.Response regulator genes expression of S.suis 2 after PMN stimulationExtraction of total bacterial RNA after PMN stimulus for 3 h and detected the 15response regulator genes expression using qRT-PCR.This experiment discovered that the vraR（SC84₀373）,ciaR,SC84₁224,SC84₁439和SC84₁522 gene transcription levels were increased significantly on exposure to PMNs compared with SC19 incubation alone,whereas the other response regulators showed little or no response to PMN stimulation.3.The Role of VraSR in innate immune evasionFurther PMN-mediated killing assay was used to identify the resistant ability of SC19,ΔvraSR and CΔvraSR to human PMN.It demonstrated that the deletion of VraSR affect the ability of S.suis 2 to survive in PMN.Furthermore,flowcytometry was used to detect the phagocytosis of SC19,ΔvraSR and CΔvraSR by PMN.Along with the oxidative（H₂O₂）and non-oxidative（Lysosome）microbicidal assays in vitro,it all demonstrated an increased sensitivity of S.suis 2 to PMN after VraSR deletion.4.Regulation of S.suis 2 capsule-synthesis and cell adherence by VraSRThe capsule of S.suis 2 has an influence on the phagocytosis of PMN,so we evaluated the thickness of SC19,ΔvraSR and CΔvraSR through transmission electron microscopy（TEM）and compared the difference of sialic acid level as well as capsule-associated genes expression.We found that mutant showed a reduction in adherence to HBMEC cells accompanied by a thinner capsule,which was contradictory to previous studies showing that non-encapsulated bacteria exhibited higher adherence to cells.We hypothesized that the absence of VraSR may have a more significant effect on adherence than the thinner capsule.These findings were in accordance with the down-regulation of a series of reported adhesion-associated genes by qRT-PCR.5.The research of VraSR on pathogenicity and inflammatory responsesBALB/c as a good model,survival curves and bacteria burden on various tissues demonstrated that the deletion of VraSR has diminished virulence and can be eliminated more easily in host tissue.In addition,VraSR was also found no influence on the production of many inflammatory cytokines at an early stage of S.suis 2infection but then sharply decreased compared with SC19.