Analysis Of Functional Domain And The Antibacterial Activity Of Streptococcus Suis Phage Lysin Ply5218 In Vitro And In Vivo

Streptococcus suis is a zoonotic pathogen infecting piglets and causing arthritis,septicemia,and meningitis,which poses a very serious threat to pig industry and public health.So far,the prevention and control of Streptococcus suis rely mainly on antibiotic treatment,however,the abuse of antibiotics leads to serious resistance and multi-drug resistance characteristics from most of clinical pathogenic isolations.The problem of bacterial resistance is worsening,which hinders the effective of the disease control.Therefore,it is particularly important to seek new antimicrobial agents.Lysin is a potential new antibacterial agent with high efficiency,rapid sterilization,strong specificity,and low resistance.In this study,based on the good effect of Streptococcus suis type 9?SS9?bacteriophage lyase Ply5218 treatment in vitro and in mice,Ply5218was used to treat the piglet disease model.A 20kg piglet neck-challenge/lysin-treatment model was used to evaluate the in vivo anti-infection effect of Ply5218.Sixteen piglets were divided into four groups:untreated group,untreated group,immediate challenge group and delayed treatment group as for 1 h after challenge.The challenged piglets were injected with 10 mL(1.4×10¹⁰ CFU/mL)SS9 virulent strain HA9801,and then injected with 10 mL?512 U/mL?lysin Ply5218 or PBS immediately.The delayed treatment group which is treated one hour after the challenge,treated with 10 mL?512 U/mL?of lysin Ply5218 injected into the neck.The survival status,clinical signs,and body temperature were recorded.The blood bacterial loads and proinflammatory cytokine levels in the blood were tested right after the study.The results showed that injection of lysin Ply5218 immediately after challenge could effectively control Streptococcus suis infection in piglets.The survival rate of the immediate treatment group reached 75%,while the survival rate of the control group was 0%.The temperature of surviving piglets returned to normal gradually.The bacterial loads decreased gradually;Within 3 days of immediate treatment,the expression level of proinflammatory cytokines in the treatment group was significantly lower than those of the untreated group,and the health status of the piglets immediately after treatment was gradually restored to the normal level.In order to make better use of lysin Ply5218 in clinical treatment,we optimized the codon of Ply5218.The optimized lyase gene fragment was cloned into the eukaryotic expression vector PPlczA.The expression was induced and the expression condition was then optimized in X₃₃ yeast competent cells.The results showed that the optimal conditions for the expression of Ply5218 in yeast were as follows:28? for 48 h?1% methanol was added every 24 h?.After the intraperitoneal injection of HA9801,the ICR mice were treated with X₃₃-Ply5218(Ply5218 was expressed in yeast cell X₃₃),Ply5218 protein,and X₃₃ yeast cells Control and PBS buffer control oral treatment,respectively.At 24 h,48 h,72 h,and 96 h post infection bacterial loads were measured in each group of mice blood,liver,and spleen.The titers of bacteria in mice orally administered with X₃₃-Ply5218 yeast cell group and Ply5218 protein group,were significantly reduced compared to non-treated group,indicating that oral administration of mice X₃₃-Ply5218 and Ply5218 can effectively treat Streptococcus suis induced infection in mice.To further identify the minimal active domain and the key amino acid of Streptococcus suis type 9 bacteriophage lysin Ply5218,the full-length lysin Ply5218 and the catalytic domain(Ply5218_1-147)were truncated and the key amino acids that may be involved in the activity were site-directed mutated,to study effect of the truncation and mutation to the enzyme cleavage activity of each lysin.The minimum active domain and key amino acids of Ply5218 were determined by comparison with the activity of the full-length lysin.The results showed that the truncated fragment Ply5218_1-147 could form fusiform circle on the plate and still maintain the activity of lysing bacteria,while the truncated fragment shorter than Ply5218_1-147 lost the lysing activity.It is elucidated that Ply5218_1-147 is the minimum active domain of Ply5218.At the 8th,58th,136th,and 142th amino acid residues,the activity of the cleavage activaty decreased.After amino acid mutations at positions 34 and 144,the cleavage activity was completely lost.In summary,in piglet treatment study,injection of Ply5218immediately after challenge can effectively control Streptococcus suis infection;after codon optimization,lysin Ply5218 can be expressed in yeast cell X₃₃.Oral administration of X₃₃-Ply5218 yeast cells provided protections to mice after challenge with HA9801.Through truncation and site-directed mutagenesis,Ply5218_1-147 was found to be the minimal function domain of Ply5218,and six key amino acid related to cleavage activity were identified.The results provided data for further revealing of the cleavage mechanism and optimization of Ply5218.