The Mining Of Ply5218 :A Novel Prophage Lysin Habored In Streptococcus Suis

Streptococcus suis?S.suis?is an important porcine bacterial pathogen and emerging zoonotic agent that causes meningitis,sepsis and arthritis in piglet or meningitis in human.A great loss in the swine industry were responsible for the outbreaks of S.suis infection.The main and conventional control measures for S.suis infections are antibiotics.However,the global emergence of multidr?g-resistant?MDR?bacteria is an increasing threat to public health worldwide.Alternative antibacterial agents with extended ability for curing some infections caused by resistance bacteria need to be developed.Bacteriophage lysins,the weapon of phages to digest the host bacterial cell wall for the release of the progeny phages has sparked an interest in a promising therapy.In this work,based on the genomic information analysis on GeneBank,an endolysin Ply5218 was evaluated in a prophage named phi5218 harbored in SS9 genome on the GeneBank.The analysis showd that Ply5218 have a promising ability for curing S.suis infections.The recombinant plasmid pET28a?+?-Ply5218 was constructed.After confirmation by sequencing,the correct plasmids were transformed into the E.coli BL21?DE3?for expression.The purified His-tagged Ply5218protein was approximately 35 kDa when determined on the SDS-PAGE.In vitro,the results showed that 34 out of 36 tested S.suis strains could be lysed by Ply5218,only three S.suis strains could not be lysed.The minimum inhibitory concentration?MIC?of Ply5218 was 2.0?g/mL against S.suis strain HA9801,an activity many times greater than the lysins reported previously.Ply5218 at 10?g/mL in vitro exerted broad antibacterial activities against S.suis strain HA9801 OD₆₀₀ ratios decreased from 1 to 0.2 within 15min.Moreover,Ply5218 showed favorable thermal stability.It was stable at 50?>30 min,4?>30 days,-80?>1 year.By addition of Ca²⁺,the activity of Ply5218 on HA9801 is improved.Ply5218 prefered high pH environment.The stable lytic activity with wide range of S.suis stains makes it possible for clinical therapy.In vivo,mice were injected with 7×10⁹ CFU of S.suis HA9801 and treated 0.2 mg Ply5218?10 mice?or 0.2 ml of PBS?10 mice in parallel?2h later.Nine of 10 mice of the control group were dead within 36 h,while90%?9/10?mice of the treatment group remained alive.Finally,Ply5218maintained high antibacterial activity in some bio-matrices,such as culture media and milk.The data indicate that Ply5218 has the characteristics to be an effective therapeutic agent against S.suis infections.