Streptococcus Suis Uptakes Carbohydrate Source From Host Glycoproteins By N-glycans Degradation System For Optimal Survival And Full Virulence During Infection

Streptococcus suis is an important zoonotic pathogen that can cause clinical symptoms such as human and swine sepsis,meningitis,endocarditis,arthritis and pneumonia.It also poses a major threat to human health and public health security.S.suis can be divided into 33 recognized serotypes based on the differences in capsular polysaccharide surface antigens,of which S.suis type 2（SS2）is a virulent type.At present,although some progress has been made in the study of SS2’s cell adhesion,biofilm formation,toxicity,and stress response during infection,the mechanism of SS2’s survival and proliferation in the host’s blood and causing sepsis is still unclear and remains to be determined explore further.1.Bioinformatics analysis of endoSS-related insertion sequences in SS2 ZY05719In the early stage,the laboratory conducted a molecular epidemiological investigation of 101 S.suis strains through bioinformatics,and identified six specific genes epf,sly,rgg,endoSS,SMU₆1-like and SpyM3₀908.These genes only exist In strong strains rather than weak strains.A further comparison revealed that most of these genes were located within the approximately 2 Kb insertion region of the ZY05719 genome,and only endoSS was located above the 20 Kb insertion region.This region is functionally speculated as a N-glycan degradation system that contains a glycosyl transporter and related glycosyl hydrolase.Comparing this region with genes related to Streptococcus pneumoniae TIGR4,it was found that the homology was higher,reaching a maximum of 70%.According to related reports of S.pneumoniae,the gh92,endoD,and ABC transport systems in this area have certain effects on the growth,proliferation,and virulence of bacteria.Transcriptome analysis showed that the endoSS-associated region genes had significantly increased transcription levels in vivo.These results imply that endoSS-related insertion sequences are related to SS2 survival,immune evasion,and virulence,which deserve our attention.In this study,gh92 and endoSS genes were selected for further exploration.2.Construction of endoSS and gh92 deletion strains and complementary strains and their effects on SS2 serum growth and virulenceUsing the SS2 virulent strain ZY05719 as the parental strain,endoSS,gh92 deletion and complementary strains ΔendoSS,CΔendoSS,Δgh92,and CΔgh92 were constructed by homologous recombination,respectively.The results of the serum growth test showed that,in both inactivated and non-inactivated serum,compared with the other four strains ofΔendoSS,they showed obvious growth defects（**P<0.01）,but did not show growth differences under THB culture..The zebrafish LD50 assay and mouse survival experiments showed that ΔendoSS and Δgh92 were significantly less toxic than CΔendoSS,CΔgh92,and ZY05719.The organ distribution results showed that in the brain,blood,spleen,and kidney organs of mice,the amount of colony bacteria in the deleted strain was significantly lower than that of the parent strain and the complementary strain（***P<0.001）.Therefore,endoSS and gh92 genes have a greater effect on SS2 virulence.3.EndoSS and GH92 proteins can synergistically degrade RNase B high mannose glycoprotein to maintain SS2 growthThrough the evolutionary tree,we found that the endo-β-N-acetylglucosaminidase（ENGase）family can be divided into two families,GH18 and GH85.Among them,EndoSS of SS2 and EndoD（GH85）of S.pneumoniae have high homology.This protein can degrade the high-mannose N-glycan glycoprotein in the host by cleaving the β-1,4 glycosidic bond,ingest and utilize the monosaccharide sugar source,and maintain its own survival and colonization,which is different from EndoS（GH18）of Streptococcus pyogenes.Express and purify EnodSS protein and GH92 protein respectively,and incubate with IgG and RNase B.EndoSS and GH92 can synergistically degrade RNase B,but not degradable IgG;in the serum growth test,add hydrolyzed RNase B and fetuin,ΔendoSS growth back to normal.CDM culture uses glucose and fetuin as the only carbon sources.Both ΔendoSS and Δgh92 strains can grow on CDM medium with added fetuin,but ΔendoSS cannot achieve the same cell density as wild-type and Δgh92 strains.There was no significant difference in growth of all strains on CDM medium containing glucose.In summary,this study proves that the endoSS-related insertion sequence in SS2 belongs to the GH85 family in ENGase,and plays the role of the N-glycan degradation system.It can cut the host’s high-mannose-type glycoprotein to take up a carbon source,thereby keeping it in the blood expression of growth and virulence.