| Klebsiella Pneumoniae(KP)belongs to Enterobacteriaceae,which can infect human and many kinds of animals,posing a great threat to public health and animal breeding industry.Currently,antibiotics are commonly used to treat K.pneumoniae infections,and their widespread use has led to the development of multidrug-resistant bacteria.Under the threat of multidrug-resistant bacteria,especially pan-drug-resistant bacteria,bacteriophages and their derivatives have been reemphasized and applied to prevent and control K.pneumoniae infection.In this study,169 strains of K.pneumoniae phage were isolated from sewage,and a small library of K.pneumoniae phage was established.Candidate phages were identified by phage genome analysis and basic biological characteristics analysis.Meanwhile,K.pneumoniae phage was found to have capsular type specificity.Tail fiber protein(TFP)replacement confirmed that the host specificity of K.pneumoniae phage was related to the TFP gene,and further locked the host range determination region.Besides,the capsule of K.pneumoniae is one of the host receptors of phage P560.Phage-derived depolymerase was found to have polysaccharides-degrading activity against capsular polysaccharides(CPS)of KL47 and KL64 KP respectively,and its antibacterial effect was also proved in vitro and in vivo.Three-dimensional structure of KL47 depolymerase P560dep was analyzed,and the catalytic domain and key catalytic amino acid sites were identified.This study provides a theoretical basis for customizing phages with synthetic biology and developing novel depolymerases.1 Isolation and characterization of K.pneumoniae phageCollection of K.pneumoniae and phage isolation are the premise of phage related researches.Therefore,this study firstly carried out isolation and identification,basic biological characteristics analysis and whole genome sequencing analysis of K.pneumoniae lytic phages.A total of 169 strains of KP phages were isolated from domestic sewage and medical sewage,8 of which were observed by transmission electron microscopy,and 17 phage genomes of which were extracted,sequenced and analyzed.The results showed that 5 strains belonged to Przondovirus.Host range analysis of the phage showed that KP phage had KL-type specificity.We selected P560,P509 and P545 from the lytic phages for basic biological characteristics analysis,providing new candidate phages for the bank of K.pneumoniae phage and candidate phages with good characteristics for phage therapy.2 Identification and modification of host range-determining protein of phagePhages are a promising antibacterial agent because of their specificity,but they are also limited by their narrow host range.In order to reveal the molecular basis of KP phage host range formation and apply synthetic biology to recustomize genetically engineered phages,this study used traditional phage homologous recombination technology.Genetically engineered phages RPA3 and RPB1 were obtained by replacing the TFP genes(ORF42 and ORF43)in phage P560 with the TFP gene(ORF38)in phage P510,respectively.RPA3 and RPB1 completely obtained the host range of P510,only by changing TFP gene.Engineered phage RPA3 not only had significant bacteriostasis effect on planktic CRKP Kp30,but also had significant inhibitory effect on the formation of biofilm and the removal of mature biofilm.Our study revealed that TFP protein is an important determinant of KP phage host-specific formation and suggested that engineered phages may be an effective approach for phage therapy in the future.In addition,the phage cocktail JS-KPP3 developed by us can effectively inhibit pan-resistant KL47 and KL64 CRKP,which has the potential as an environmental antimicrobials.JS-KPP3 phage cocktail is expected to be widely used in breeding industry and clinical environment purification with the continuous optimization of the component of JS-KPP3 phage cocktail.3 Studies on phage-resistant bacteria and host receptorThe host receptors of phages included LPS,outer membrane proteins,capsules,flagella and lipids.In order to identify host receptors and reveal the mechanism of phage resistance,30 Kp42 mutants resistant to phage P560 were firstly isolated,and the wbaP mutants Kp42-P560R was identified by PCR and sequencing analysis.Then,the whole genome sequencing and comparative genomic analysis of the wild strain Kp42 and the phage-resistant mutants Kp42-P560R showed that no mutation of outer membrane protein was found,however,insertion mutation of wbaP gene affecting the formation of CPS,which caused translation frameshift.To verify that the capsule is the host receptor of phage,we constructed the deletion mutant Kp42:ΔwbaP and the complementary strain Kp42 pwbaP,and found that the wbaP gene affected the formation of capsule,while capsule affects the lysis ability and adsorption capacity of phage P560 to it.This study have shown that capsule of K.pneumoniae Kp42 participated in the adsorption of phage P560,and was one of the essential receptors for phage P560 to infect Kp42 strain.The study of phage resistance mechanism and host receptor laid a theoretical foundation for the application of phage therapy.4 Expression,identification and antibacterial study of recombinant depolymerasePhage-derived antimicrobial proteins mainly include lysin,depolymerase,holin,transport signal polypeptide and transmembrane peptide,etc.We predicted two phage-derived depolymerases P560dep and P510dep,targeting KL47 and KL64 K.pneumoniae capsular polysaccharides,respectively,and conducted prokaryotic expression and purification,enzyme activity analysis and specificity analysis of recombinant depolymerases,and evaluated their antibacterial effect in vitro and in vivo.The results showed that depolymerase P560dep can degrade the CPS on the surface of KL47 K.pneumoniae,and the depolymerization spectrum of P560dep was the same as the host range of phage P560.P510dep can degrade KL64 CPS,and the depolymerization spectrum matches the host range of P510.Both depolymerase P560dep and P510dep significantly inhibited biofilm formation.In vivo experiments,P560dep can prevent and treat bacteremia infection caused by carbapenem-resistant K.pneumoniae in mouse models.Before and after KL47 CRKP infection,intraperitoneal injection of a single dose of depolymerase(50 μg/mouse)can protect 90%-100%of the mice and reduce the pathological changes of lung and liver.Therefore,depolymerase was an attractive antibacterial agent and can be used as an antimicrobial therapy schedule.5 Study on structure and function of depolymerase P560depAt present,there are many researches on antibacterial activity of depolymerases,but few researches on structure of depolymerases.In order to reveal the relationship between structure and function of depolymerase,3D structure of depolymerase P560dep was obtained by crystal diffraction method and selenomethionine method with a resolution of 1.6 ?.It consists of four domains:N-terminal domain,catalytic domain,lectin-like domain and C-terminal domain.Compared with other depolymerases,P560dep has a novel modular structure in which two tandem lectin-like domains are inserted into its dextral parallel β helix domain.Site-directed mutations were carried out on 18 amino acid sites in the 6 predicted candidate catalytic domains,and enzyme activity tests showed that 4 regions and 6 residues played a role in the catalytic function,among which residues Asp298,Asp311 and Glu314 in region 5 and Glu554 in region 6 played a major role in the catalytic function.At present,the existence of multiple catalytic pockets in a single tail fiber protein has not been reported.Hydrophobic pockets in the lectin-like domain may promote the ability to bind to polysaccharides.The analysis of the structure and function of high-efficiency depolymerase with structural framework provides a theoretical basis for further design of broad-spectrum enzyme against K.pneumoniae.In conclusion,aiming at the serious problem of drug resistance of K.pneumoniae,this study established a small K.pneumoniae phage library,and identified candidate phages for phage therapy through biological characterization and genome sequence analysis.The host specificity of K.pneumoniae phage was confirmed to be related to the TFP gene by gene replacement.The phage-resistance mechanism based on wbaP gene mutation was demonstrated by phage resistance analysis and gene deletion analysis,and the capsule of K.pneumoniae was proved to be an important host receptor for phage P560.In this study,phage-derived depolymerases targeting KL47 and KL64-type capsules were also found,demonstrating their antibacterial effects in vitro and in vivo;the homotrimeric structure of KL47-type depolymerase P560dep was analyzed,and 6 key amino acid catalytic sites of two catalytic domains.This study provides a K.pneumoniae phage candidates for phage application,and also provides a theoretical basis for the use of synthetic biology to customize phage and develop novel depolymerases. |
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