Research On The Mechanism Of Heparin-Binding Protein Releasing Caused By Streptococcus Suis Serotype 2 Chinese Isolate

Streptococcus suis (SS) is one of the most important zoonotic pathogens. Among the 35 known serotypes, SS2 is the most prevalent serotype, and its pathogenicity is the doughtiest with the hallmarks of meningitis, endocarditis, arthritis, septicaemia, endophthalmitis and permanent hearing loss etc. In 1998 and 2005, S.suis outbreaks twice in humans which occurred in Jiangsu and Sichuan of China, respectively, inhibiting a much higher human mortality. A majority of the fatal cases represent typical symptoms of strep tococcal toxic shock syndrome (STSS) that has not be found in foreign countries, and few reported in domestic. Nevertheless, at present the reports of toxic shock syndrome is mainly focused on Staphylococcus aureus, while reports of STSS is mainly focused on Group A Streptococcus, the pathogenic mechanisms of toxic shock syndrome in SS remain little understood.In prior study, compared the HBP level of the serum from 8 meningitis patients, 6 STSS patients and 14 healthy adults, found that the average HBP level of TSS patients was remarkably higher than that of meningitis patients and healthy people, indicate that higher HBP level in serum is the immediate cause of TSS. Based on these findings, our laboratory early work on the screen of SS HBP stimulating factor, and preliminarily identified the stimulating factor is Streptococcus suis hemolysin (suilysin, SLY). On this basis, SLY is the stimulating factor for HBP was further verified and the molecular mechanism of HBP release was preliminary explored. The main results are as follows.1 Validate SLY is stimulating factor of HBP releasingUsing saturated ammonium sulfate precipitation, combined anion chromategr-aphy and hydrophobic chromatography, we extracted nature SLY (nSLY) from culture supernatants of SS2. Molecular biology methods was used to obtain recombinant SLY. Using anti-(recombinant) SLYpolyclonal antibody, SLY gene knockout strains (ASLY) was analyzed by western blot and the result showed SLY protein was not expressed in ASLY. The ability to stimulate whole blood and PMN(poly-morphonuclear nuetrophil) which isolated from whole blood to release HBP was evaluated by comparing nSLY, rSLY and culture supernatants of 05ZYH33 (wild type) and ΔSLY (isogenic non-suilysin mutant).The results showed that when the concentration of NSLY was 1μg/mL, it can stimulate human whole blood and PMN to release more than 30% HBP of total storage; but 5μg/mL P353V protein still can not stimulate HBP to release; compared with the stronger ability of 05ZYH33 to stimulate HBP release, this ability of ΔSLY supernatant was almost completely lost. These results indicate that SLY is the stimulating factor of HBP release in SS.2 Explore the pattern of HBP releasingSLY above a certain concentration can cause severe cracking of PMN, and PMN will be killed. But through detecting PMN activity indicators -superoxide anion release, found that 1μg/mL nSLY can not cause significant effect in cell vitality of PMN.The results indicated that the release of HBP was occurred when PMN maintain physiological activity. To validate whether the release pattern of HBP was degranulation, the expression of target CD antigen molecules (CD11b, CD66b, CD63)on the surface of PMN was detected by FCM(flow cytometer). The results showed that two of the three target CD antigen molecules (CD66b and CD63) had a high expression on the cell surface. Furthermore, scanning electron microscopy was used to observe the cell surface blebbing, found that after the stimulation of nSLY, the cell surface of PMN appear a large number of vesicle’s protrusions. The vesicle’s protrusion is a performance of intracellular protein particles fusion on the membrane. Comprehensive these results, we confirmed that the release of the HBP was conducted by PMN degranulation.3 Explore the signaling pathway of HBP releasingPMN degranulation involves a variety of signaling pathways, in order to explore the pathway of HBP releasing, a variety of different signaling pathway specific inhibitors was used to suppress HBP release respectively. The results showed that the specific inhibitor of phosphatidylinositol 3-kinase(PI3K)pathway and p38 mitogen-activated protein kinases (p38 MAPK) pathway have significant effects. Simultaneously, the release of a small molecule Leukotriene B4 (LTB4) increased by stimulating with nSLY, and its receptor LTB4-BLT1 specific inhibitor U-75302 had a significant inhibitory effect. According to these results, we presumed that the signaling pathway related to HBP release were PI3K and p38MAPK, and the related receptor was LTB4-BLT1.