Polysaccharide Depolymerase Encoded By The Phage SH-KP152226 Confers Specific Activity Against Multidrug-resistanct Klebsiella Pneumoniae

Klebsiella pneumoniae is an opportunistic bacterial pathogen that causes a wide range of nosocomial infections.It widely colonizes in human mouth,skin,respiratory tract,and digestive tract under normal physiological conditions.K.pneumoniae can become pathogenic in patients with compromised immune systems,causing various diseases such as urinary tract infections,pyogenic liver abscess(PLA),and pneumonia.The increasing prevalence of carbapenem-resistant K.pneumoniae(CRKP)has led to the loss of the effectiveness of antibiotics against such infections.Thus,the Centers for Disease Control(CDC)of the USA designated K.pneumoniae as an urgent threat to public health.Therefore,it is necessary to develop new effective treatments against K.pneumoniae.Bacteriophages are viruses that naturally attack bacteria.However,due to the presence of EPS matrix at the cell surface,activity of phage in infecting bacteria is often hindered.Interestingly,during coevolution with their hosts,the phages have produced a variety of polysaccharide depolymerases that degrade the polysaccharide compounds such as EPS and CPS in order to bind to the surface receptor of bacteria.In previously published studies,various depolymerases have been proposed as adjuvants to combat infections of drug-resistant bacteria by preventing biofilm formation or degradation of mature biofilms.An epidemiological study found that the predominant capsular type of CRKP strains in China is the K47 serotype.Thus,we isolated,characterized,and sequenced a K.pneumoniae bacteriophage(SH-KP152226)that specifically infects and lyses K.pneumoniae capsular type K47.Whole-genome sequencing demonstrated that SH-KP152226 contains a genome of 41,420 bp that encodes 48 predicted proteins.Among these encoded proteins,Dep42,the gene product of ORF42,is a putative tail fiber protein.Bioinformatics analysis shows that its central region(residues 300-599)has a β-helical structure pectate＿lyase＿3＿,which has hydrolyzed pectin active.Therefore,it is predicted that it may be a polysaccharide depolymerase.We cloned the tail fiber protein gene(ORF42)of phage SH-KP152226 into the expression vector p SUMO3,constructed the recombinant plasmid p SUMO3-Dep42,and purified the Dep42 protein.We demonstrated that recombinant Dep42 showed specific enzymatic activities in the depolymerization of the K47 capsule of K.pneumoniae and was able to significantly inhibit biofilm formation and/or degrade formed biofilms.We also showed that Dep42 could enhance polymyxin activity against CRKP biofilms when used in combination with antibiotics.In conclusion,these results indicate that combination of the identified novel depolymerase Dep42,encoded by the phage SH-KP152226,with antibiotics can effectively combat the formation of CRKP biofilms and has potential clinical application value.