The Interaction Between Suilysin And Fucose On The Erythrocyte Membrane And Immunization In Piglet

Streptococcus suis is an important zoonotic bacteria that causes meningitis, arthritis,myocarditis and streptococcal toxic-shock-like syndrome（STSLS）.Streptococcus suis disease were erupted on the year of 1998 and 2005,causing significantly higher severe streptococcal toxic-shock-like syndrome than other country in the world. Suilysin（SLY）presented as the only secretory protein toxin,resulting in streptococcus suis streptococcal toxic shock syndrome, erythrocyte hemolysis and stimulating the inflammatory cytokines release from macrophages and severe inflammatory reaction in experimental animals.Non-toxic SLY mutations, a kind of protective antigen,could stimulate immune protection in mice.SLY belongs to Cholesterol dependent cytolysins（CDCs）, possessing the CDCs typical molecular crystal structure.Perfringolysin O（PFO） was the prototype for all CDCs. CDCs could bind to membrane cholesterol and form ring oligomer complex, resulting the erythrocyte hemolysis. However,in recent years,studies involved in interaction between Intermedilysin（ILY）, Vaginolysin（VLY）, Pneumolysin（PLY） and membrane broke the assertion that cholesterol was the unique CDCs receptor.The study found that ILY, VLY specifically bind human CD59 molecule, PLY and Lewis X has a higher affinity Kd value of 1.88 × 10-5M.Since SLY^P353L mutant could no longer cause erythrocyte hemolysis, but erythrocyte agglutination.Cholesterol could not completely block the SLY^P353L binding to erythrocyte membrane.So, SLY being similar to ILY and VLY existed multiple binding sites between the red blood cells. This study analyzed the SLY interaction with monosaccharide and rSLY^P353Lsubunit vaccine Immunization in Piglet,results are as follows:1. rSLY, rSLY^P353L presenting on erythrocyte membrane in different formsrSLY, rSLY^P353L were incubated with MRBC（ Mouse Red Blood Cells）suspension. The rSLY membrane complexes were collected by centrifugation since rSLY completely lysed the MRBC（Mouse Red Blood Cells）. MRBCs were pelleted and lysed with hypotonic buffer after rSLY^P353L incubation. The rSLY^P353L membrane complexes were also collected by centrifugation. The membrane complex were detectedthrough SDS-AGE gel electrophoresis and western blotting(primary antibodies rSLY^P353L Ig G). Monomer, dimer and oligomer were observed on the rSLY membrane.In contrast, rSLY^P353L was observed only as a monomer.2. Monosaccharide influence hemagglutination of rSLY^P353L and hemolytic activity of rSLYAccording to the above results and article reports,SLY may bind to other receptor,particularly glycosylation component, beyond membrane cholesterol. Fucose, Mannose and Glucose were utilized for blocking: the blocking efficiency was closely related to monosaccharide concentration, 278 mmol/L monosaccharide could significantly block 2HU（Hemolytic Unit） rSLY or 2HAU（Hemagglutination Unit） rSLY^P353L, but 70mmol/L failed. 278 mmol/L sugar coudn’t to block hemolytic activity of 16 HU rSLY or agglutination effect of 16 HAU rSLY^P353L. Testing with fucose-depleted MRBC, the hemolytic effect of 2 HU rSLY was observed as slightly decreased and the agglutination titer of rSLY^P353L preparation decreased by two-fold compared with the MRBC treated with the buffer.3. Cholesterol and fucose blocking rSLY and rSLY^P353L binding abilityrSLY and rSLY^P353L were pre-incubate with cholesterol and fucose, before the erythrocyte addition. MRBCs were pelleted and lysed with hypotonic buffer to collect membrane comple after fully incubation.The membrane complex were detected through SDS-PAGE gel electrophoresis and western bolting. The result showed that the blocking efficiency of cholesterol was better than fucose, and cholesterol together with fucose could block rSLY binding to membrane and even completely block rSLY^P353L binding to membrane.4. Native-polyacrylamide gel affinity measurement for rSLY rSLY^P353L and monosaccharideThe separating gel contained 12%（v/v） polyacrylamide, and 110 mmol/L monosaccharide. The solutions of BSA, rSLY, and rSLY^P353L were each mixed with native loading buffer. Electrophoresis was performed under ice water mixture to maintain its activity. The mobility ratio of rSLY and rSLY^P353L decreased by 1.7% and2.7%, respectively, no changes were observed in gel with mannose and glucose.5.Molecule docking analysisThe crystal structure data of rSLY was download from NCBI and data of fucose from Zinc. The best binding pocket and the optimal interaction model sores were calculated by CLC Drug Discovery Workbench 2. Fucose may bind to the first Domain1 of SLY and forming hydrogen bonds with Tyr 98, Pro 99, and Val 150.6. Immunization with rSLY^P353L in PigletSuilysin subunit vaccine, prepared with aluminum hydroxide gel as adjuvants, is divided into three groups for immunization: inoculating 50 ug rSLY^P353L each time for two times, inoculating 150 ug rSLY^P353L each time for two times, inoculating 500 μg rSLY^P353L just once. Enzyme linked immunosorbent test（ELISA） being used for the detection of immune serum antibody titer. Accordingly, a high dose of disposable inoculation group can not detect antibody, twice vaccinated group,partially,were able to produce specific antibodies which following a different dynamic cures.The titer can be as high as 1:1024.In a word,suilysin can interact with membrane fucose, suilysin subunit vaccine, prepared with aluminum hydroxide gel,was able to be used for piglet immunization.