Expression,Purification And Antibacterial Activity Of Klebsiella Pneumoniae Phage Endolysin PlyKp3 In Vitro

Objective:Infection is the primary reason leads to burn death.Klebsiella pneumoniae is one of the main pathogenic bacteria causing burn infection in recent years,and the prevalence of Carbapenem-resistant Klebsiella pneumoniae(CRKP)has brought severe challenges to the efficacy of traditional antibiotics.Endolysins are enzymes encoded by bacteriophage that can lyse bacterial by hydrolyzing the cell wall of bacteria,which provide a potential solution for the treatment of resistant bacterial infections.In order to provide a new treatment for Klebsiella pneumoniae infection,also provide experimental and theoretical basis for the development of PlyKp3 as an antibacterial drug,we expressed and purified Klebsiella pneumoniae bacteriophage endolysin PlyKp3,which was isolated from a lytic phage Kpssk3 targeting a clinical Klebsiella pneumoniae Kp3,and verified its antibacterial activityMethods:1、Expression and purification of Klebsiella pneumoniae phage endolysin PlyKp3The ORF15 gene of Klebsiella pneumoniae bacteriophage was cloned into expression vector pET28a and further transformed into E.coli for amplification.The recombinant plasmid was induced by IPTG and expressed.PlyKp3 was purified by Ni-NTA2、To detect the In vitro lysis effect of Endolysin PlyKp3 on host bacterium kp3PlyKp3 was incubated with the host strain Kp3.The changes of OD600 before and after PlyKp3 addition were counted by turbidimetric assay,the changes of the number of viable Kp3 after PlyKp3 addition were measured by bacterial counting method,and the morphological differences of Kp3 before and after PlyKp3 addition were observed under light microscope and transmission electron microscope3、To detect In vitro lysis effect of PlyKp3 on CRKP clinical isolatesPlyKp3 was incubated with 21 clinical isolates of CRKP overnight.The changes of OD600 before and after adding PlyKp3 were analyzed by turbidimetric assay,so as to evaluate the in vitro lysis effect of PlyKp3 on CRKP clinical isolates4、To detect the split bacterial spectrum of Endolysin PlyKp3PlyKp3 was incubated with Acinetobacter baumannii,Pseudomonas aeruginosa,Escherichia coli and Staphylococcus aureus for 30 minutes respectively.The changes of OD600 before and after adding PlyKp3 were counted by turbidimetric assay to evaluate the split bacterial spectrum of PlyKp3Results:1、PlyKp3 was expressed and purified in vitro.PlyKp3 with the size of 18 kDa,purity of 95%and concentration of 2mg/mL was obtained2、After Kp3 was co incubated with PlyKp3 at the final concentration of 2mg/mL,the OD600 value decreased from 2.5 to 0.5,the number of viable bacteria decreased from 1.0×109CFU/mL to 4.0×108CFU/mL,and the original morphology of bacteria disappeared under light microscope and transmission electron microscope3、After 21 CRKP clinical isolates(different types)were co incubated with the final concentration of 2mg/mL PlyKp3 overnight,the difference of OD600 value between the two groups was statistically significant(P<0.05)4、The OD600 values of Gram-negative bacteria including Acinetobacter baumannii,Pseudomonas aeruginosa and Escherichia coli incubated with PlyKp3 for 30 min showed statistical difference(P<0.05),while the OD600 values of Gram-positive bacteria Staphylococcus aureus showed no significant change(P>0.05)Conclusion:The results showed that the in vitro expression,purification and antibacterial activity of PlyKp3 from Klebsiella pneumoniae phage were studied.It was found that PlyKp3 not only had strong in vitro cleavage to Klebsiella pneumoniae,but also had good cleavage to A.baumannii,P.aeruginosa and E.coli,showing its antibacterial activity to Gram-negative bacteria,suggesting that it is not only a potential effective antibiotic for the treatment of Klebsiella pneumoniae infection,but also a new drug against Gram-negative bacteria expected to be.