Identification Of Key Domain And Key Amino Acid Sites Of Lysin Encoded By Phage Infecting Streptococcus Suis 2

Streptococcus. suis 2(S. suis 2) is an important zoonotic pathogen, the increasing prevalence of antibiotics resistant in S. suis 2 strains becomes one of the greatest challenges against antibiotic therapy, the phage-based antimicrobial strategy seems to be a potential way to deal with this problem. Bacteriophages are viruses that infect bacteria and release progeny bacteriophages by synthesizing lysin to lyse cell wall of host bacteria at the end of the phage lytic life cycle. Bacteriophage SMP is the only published S. suis 2 lytic phage. The lysin encoded by SMP, termed LyACCG(or LySMP), have an N-terminal amidase-5(A5) catalytic domain, two Cpl-7(C7) cell wall binging domain in the middle and a C-terminal glucosaminidase(G) catalytic domain. For identification of key domain in LyACCG and key amino acid sites in domain A5, in this research:(Part one) Identification of key domain in LyACCG:(1) Prokaryotic expression of lysin domians. Using genome of phage SMP as template, gene fragments coding LyA, LyAC, LyACC, LyG, LyCG, LyCCG and LyACCG, respectively corresponding to 1-145, 1-195, 1-240, 300-410, 195-410, 150-410 or 1-481 amino acid sites of LyACCG were amplified by PCR and inserted into pSJ2 plasmid. The recombinant plasmids were transformed respectively into E.coli BL21(DE3) for expression with 1 mmol/L IPTG induction at 27 °C. SDS-PAGE analysis showed that, all of the lysin domians were intracellular and soluble, with respective molecular weight as 19 ku, 25 ku, 30 ku, 16 ku, 28 ku, 32 ku, and 56 ku.(2) Qualitatively analysis of bacteriolytic activity of the lysin domians. Cultured cells were resuspended in buffer and sonicated in ice box. Cell debris were removed by centrifugation, supernatant were filtered to remove any remaining particles. Crude extracts of the lysin domians were used for plate lysis assay. LyA, LyAC, LyACC and LyACCG(harboring domain A5) could lyse HA9801 with the dimter of lytic ring were 1.2 cm, 1.7 cm, 2.2 cm and 1.3 cm respectively whereas LyG, LyCG and LyCCG(harboring domain G, without domain A5) did not show any lytic effect, showing that bacteriolytic activity of LyACCG attributed to domain A5 and domain G without bacteriolytic activity. LyA, LyAC, LyACC and LyACCG showed bacteriolytic activity against S. suis 2(12 strains) and S. suis 7, but they did not have any lytic activity against S. suis 9, S.equi subsp. zooepidimicus, Staphylococcus aureus(5 strains), Bacillus, E.coli and Salmonella. Bacteriolytic spectrum of LyA did not change compared with LyACCG, which implied that the specificity of LyACCG came from domain A5 and had nothing to do with domain C7 and domain G.(3) Quantitatively comparison of bacteriolytic activity between LyA, LyAC, LyACC and LyACCG. LyA, LyAC, LyACC and LyACCG were purified by Nickel ion affinity chromatography. 0.45 μmol/mL of LyACC could bring about the turbidity of HA9801 decreased by half, the concentration of LyACCG were 7 times compared with LyACC, domain G inhibited the activity of lysin; the concentration of LyA and LyAC were 29 and 17 times compared with LyACC, domain C7 played an important role when LyACC lysed HA9801, but not decisive. 2 μmol/m L of LyACC and LyACCG could bring about the density of HA9801 in biofilm decreased by 44.23% and 14.44%, which implied that domain G restrained the biofilm scavenging capacity of lysin.(4) Biological activity analysis of LyACC. LyACC exerted efficient lysis activity at pH=8. Zn2+, Cu2+ and high concentration of Ca2+ inhibited the bacteriolytic activity of LyACC, Fe2+ and Mg2+ did not affect the bacteriolytic activity of LyACC.(Part two) Identification of the key amino acid sites in domain A5.(1) Analysis of conservative level of each amino acid site in domain A5. Conservative level of each amino acid site in domain A5 was analyzed and calculated based on NCBI, Pfam and Uniport databases, eleven highly conserved amino acid sites(Y18, D30, C31, S32, G43, T53, G85, G92, G95, H96 and I107) and three less conserved amino acid sites(E70, D73 and D75) were screened out for substitution.(2) Mutation of the highly conserved amino acid sites in domain A5. Using pSJ2-lyacc as template, codons of eleven highly conserved amino acid sites were substituted with alanine(A) by PCR-based point mutation technology, respectively. After success of substitution were determined by sequencing, the mutated plasmids were transformed respectively into E.coli BL21(DE3) for expression with 1 mmol/L IPTG induction at 27 °C, expressed proteins named as Y18 A, D30 A, C31 A, S32 A, G43 A, T53 A, G85 A, G92 A, G95 A, H96 A and I107 A respectively. SDS-PAGE analysis showed that, all of them were intracellular and soluble with molecular weight as 30 ku.(3) Mutation of the less conserved amino acids in domain A5. Using pSJ2-lyacc as template, codons of three less conserved acidic amino acid sites were substituted with codons of arginine by PCR-based point mutation technology at the same time. After success of substitution were determined by sequencing, the mutated plasmid was transformed into E.coli BL21(DE3) for expression with 1 mmol/L IPTG induction at 27 °C, expressed protein named as Ly7 X. SDSPAGE analysis showed that, Ly7 X was intracellular and soluble with molecular weight as 30 ku.(4) Qualitatively analysis bacteriolytic activity of the mutants. Crude extracts of the mutants were used for plate lysis assay. D30 A, C31 A, S32 A, G43 A, T53 A and H96 A completely lost their bacteriolytic activity, D30, C31, S32, G43, T53 and H96 were key amino acid sites for the bacteriolytic activity of LyACC; the diameter of lytic ring in mutants Y18 A, G85 A, G92 A, G95 A and I107 A shortened 0.9 cm, 0.8 cm, 0.3 cm, 0.5 cm and 0.3 cm respectively compared with LyACC, Y18, G85, G92, G95 and I107 played important role when LyACC lysed HA9801; the diameter of lytic ring in Ly7 X shortened 0.8 cm compared with LyACC, E70, D73 and D75 played important role when LyACC lysed HA9801.