Study On The Expression And Antibacterial Activity Of The Depolymerase From A Klebsiella Pneumoniae Lysate Phage

AIMKlebsiella pneumoniae(K.pneumoniae)is one of the common Gram-negative conditional pathogens causing hospital-acquired infections.Since 1980s,with the emergence of drug-resistant bacteria,the treatment for K.pneumoniae infections has become more difficult.The lack of available antimicrobials has prompted the development of alternative strategies for the treatment of these infections.In this study,a K.pneumoniae bacteriophage(vB_KpnP_IME321)targeting a KN1 capsular type strain,Kp409,was isolated,characterized and sequenced.The ORF42,which was identified as encoding the putative capsule depolymerase was expressed in vitro,and the bactericidal contribution assay mediated by this enzyme was performed,and the protection and safety of this phage-derived depolymerase was evaluated.METHODSK.pneumoniae Kp409 was used as indicator bacteria to isolate bacteriophages from sewage of 307 Hospital of PLA by plaque assay.The titer of bacteriophage IME321 was conformed by double-layer plate culture method,and the muhiplieity of infection(MOI)and one-step growth curve of bacteriophage IME321 was determined.The morphology of bacteriophage was observed by transmission electron microscopy with negative staining method,and the nucleic acid of bacteriophage was extracted by protease K/SDS method.Complete genome sequencing of phage IME-321 was conducted using Illumina Miseq and bioinformatics analysis was also carried out.Gene encoding for the depolymerase of phage IME321 was amplified by PCR,and then the amplification product was introduced to pEasy-BluntE1 expression vector.The expressed depolymerase was designated Dp42 and purified through the Ni-NTA column,and detected by electrophoresis on an SDS-polyacrylamide gel.The bactericidal contribution assay mediated by the phage-encoded depolymerase was employed.A mouse model of K.pneumoniae KpP409 infection was established by intraperitoneal injection.The mice of different experimental groups were injected with2×10~7 CFU and 2×10~8 CFU 30 minutes before and 30 minutes after infection respectively.The mice were injected with 50?g Dp42 expressed in vitro by intraperitoneal injection.The mortality rate of mice was observed to determine the protective effect and therapeutic effect of Dp42 on the infected mice model.RESULTSA K.pneumoniae bacteriophage(vB_KpnP_IME321)targeting a KN1 capsular type strain,Kp409,was isolated.Transmission electron microscopy observations showed that IME321 has a icosahedral head(38 nm)and a noncontractible short tail(13nm)and belongs to the Podoviridae,and the Kp32 virus of bacteriophages This bacteriophage has a latent period of 20 min and a burst size of approximately 410pfu/cell.Whole genome sequencing revealed that IME321 has a linear double-stranded genome of 39,906 bp,with a G+C content of 52.8%and a nucleotide content of 25.4%A,24.9%C,27.9%G and 21.8%T.The annotation results showed that 49 ORFs were predicted in the IME321 genome with no integrase,toxic or tRNA genes.Based on phylogenetic tree analysis of the large subunit of phage terminal enzymes and comparison with the sequence of K.pneumoniae phages published by GenBank,IME321 has the highest homology with five strains of phages isolated from the United States and Taiwan of China.BLASTp analysis showed that ORF42 in IME321 gene sequence encoded a hypothetical tail fiber protein of T7 superfamily.The ORF42 was located in 33394-35857 and was identified as encoding the putative capsule depolymerase.The enzyme expressed and purified in the Escherichia coli BL21 system namely Dp42,and the molecular weight of the protein is about 100 kDa,could depolymerize the capsular polysaccharide of Kp409 and form translucent halos on the plates.The phage-encoded depolymerase could increase the inhibitory effect of serum on the growth of bacteria in vitro.Pre-treated with Dp42 rescued 100%of mice following lethal Kp409 challenge,and administration of this enzyme after infection significantly increased survival rates of infected mice in the animal experiment.CONCLUSIONIn this study,K.pneumoniae isolated clinically was used to isolate a lysated bacteriophage(IME321)from hospital sewage.The biological identification and genomic analysis of the bacteriophage were completed.ORF42 was cloned and expressed successfully.Its bacteriostasis and synergistic bactericidal activity with serum were videntified in vitro.Dp42 has bactericidal effect and has obvious protective effect on mice model of abdominal infection.It is worth further study to provide new ideas and new methods for the prevention and treatment of drug-resistant bacterial infections.