Characterization Of Streptococcus Suis Phage SMP Lysin And Its Receptor

Streptococcus suis is an important zoonotic pathogen that causes meningitis, sepsis, arthritis, endocarditis and pneumonia in piglet or human meningitis. It is a big threaten to public hygiene and human life. Worse more, the severe antibiotics resistance of bacteria make it difficult to the prevention and treatment of this disease. In this thesis, recombinant S. suis phage lysin, named LySMP and truncated segments Lys1, Lys2, Lys3, Lys4 were expressed and purified to eliminate the clonization of bacteria. For animal test, LySMP was chosed for anti-infectious therapy of S. suis infected zebrafish.Purified active LySMP was obtained by recombinant expression in Escherichia coli. The DNA of SMP was extracted before a pair of primers was designed according to the lysin gene, designated LySMP. After amplification by PCR, the products were then inserted into a prokaryotic expression plasmid, pET-28a(+) to obtain the recombinant plasmid pET-lys. It was introduced into E. coli BL21 competent cells and induced for expression. The expression product of about 55 kDa was obtained. The zymogram testified that it could lyse host strain SS-H. The purified product of recombinant LySMP protein was 1-3mg/ml.The in vitro characterization of LySMP was analyzed. Chromatographically purified LySMP treated with 0.8% of b-mercaptoethanol showed high degrading efficiency against PMSF or lysozyme treated cells comparing to PBS washed cells. Plate assay showed an extensive lysin spectrum of lysin lysate than those of whole phage against bacteria investigated. Fifteen of seventeen strains of S. suis serotype 2 could be lysed, as well as S. suis serotype 7 and S. suis serotype 9, Streptococcus equi ssp. zooepidemicus and Staphylococcus aureus. But E. coli and Salmonella enterica were not affected. LySMP exerted efficient lysis activity at 37℃, pH 5.2. The turbidity of bacterium investigated was observed to decrease by 1.2–68%.To investigate and choose the active lytic LySMP domains, catalyse and binding segments of LySMP together was expressed in E. coli. Four domains of LySMP at amino acid position of 1-243, 1-279, 2-300, 151-443 were expressed and the recombinant proteins were named as Lys1, Lys2, Lys3, Lys4. The resulting product were about 32kDa,36kDa,39kDa,37kDa, respectively. By zymogram, it deduced that both of Lys1 and Lys2 could lyse the host strain SS2-H. However, for Lys3 and Lys4, no band were found. The purified product of Lys1 and Lys2 protein were 1-3mg/ml.Characterization of truncated Lys1, Lys2 in vitro was tested and optimun conditions for lysis were chosed. Lys1, Lys2 lysate exhibited an extensive lysin spectrum than those of whole phage against bacteria investigated. Seven of seventeen strains of S. suis serotype 2 could be lysed, as well as S. aureus. But S. suis serotype 7 and S. suis serotype 9, S. equi ssp. zooepidemicus,E. coli and S. enterica were not affected.. Purified Lys1 showed high degrading efficiency when added 0.3% ofβ-mercaptoethanol or 20mM cysteine; while purified Lys2 showed high degrading efficiency when added 0.8% ofβ-mercaptoethanol or 15mM cysteine. Lys1 exerted efficient lysis activity at 37℃, pH 5.2 and Lys2 exerted efficient lysis activity at 37℃, pH 6.8. 100mM LySMP exerted a higher degradation activity compared to Lys1 and Lys2 under the same concentration which can obtain a reduced turbidity of 33.7% verse 27.2% and 8.9%.The in vivo anti-infective effect of LySMP was tested by using zebrafish as the infection model of S. suis serotype 2. Survival rate of lysin treated groups and S. suis challenging group were compared to investigate the anti-infective effect of LySMP. Zebrafish were fist divided into seven groups for the anti-infective test of S. suis HA9801 with 30 fish in each group. Lysin treated group 1 were inoculated with 2μg LySMP in peritoneal before challenging with 50 LD50 HA9801. In group 2, 2μg LySMP and 50LD50 HA9801 were equally mixed and inoculated to zebrafish. In group 3, 2μg LySMP was adminstered post infection, and then the survival rate was calculated. PBS, LySMP controlling groups and antibiotic group were respectively inoculated with PBS, LySMP and amoxicillin. Result showed that three lysin treated groups have anti-infective effect toward HA9801 challenging, the survival rate were 70%,66.7% and 70%. For challenging group, the survival rate was 6.7%. Survival rate of antibiotic group was 96.7%, PBS and LySMP controlling groups were 100%.Different strains of S. suis were adminstered to zebrafish to evaluate the anti-infective effect of LySMP. Two SS2 stains, SS2-H and SS2-3, one SS7 strain, SH040805, one SS9 strain, SH040817 were respectively inoculated to zebrafish and for 30min after, 2μg LySMP were again inoculated for the rescue of fish. Survival rate of four groups were 90%, 93.3%, 86.7%, 60%. Result showed that LySMP could rescue fish from high mortality rate, and LySMP could be a candidate therapy agent for S. suis disease.S. suis phage SMP has a limited host rang that can only infect SS2-H and SS2-6 strains and form plague on agar plate, which limited the use of phage. To elucidate interaction of phage and its host bacteria, chemically and enzymatically treated cell wall and extracted protoplasts were tested to confirm gradients invovled in phage adsorption. In this sturdy, SS2-H cell wall were extracted and diluted to a proper concentration. Diluted cell walls were treated with SDS, proteinase K and mutanolysin respectively. The mutanolysin treated cell wall made a reduction rate of adsorption to phages to 23.1%. SS2-H protoplasts were prepared and adsorption assay was done to find no adsorption result of protoplasts with phages. Numerous saccharides, including mannose, galactose, glucose, glucosamine, N- acetylglucosamine, rhamnose and ribose, were tested. Mannose, rhamnose, glucosamine and N- acetylglucosamine could adsorb to phage with an adsorption rate of 32.3%, 41.9%, 50% and 25.8%. In reversible adsorption assay, phages were incubated with SS2-H cell walls. At intervals of 30min, 60min, 90min, dissociated phages were separated and counted to find a gradually reduction until 100% adsorption to cell wall at 90min. It demonstrated that saccharides ingredients was the receptor of SMP instead of protein and probable ingredients were peptidoglycan or teichoic acid. Mannose, rhamnose, glucosamine and N- acetylglucosamine could be the surface binding sites of SS2-H to SMP and the binding of cell walls to SMP were irreversible.