Biological Characteristics Of Phage GH-K3 And The Mechanism Of Inducing Phage Resistance In Klebsiella Pneumoniae

Klebsiella pneumoniae?K.pneumoniae?spp.are important nosocomial and community-acquired opportunistic pathogens,which cause various infections,especially highly virulent strains with K1 or K2 serotype that pose serious threats to public health and animal husbandry.Due to the abuse of antibiotics,multidrug-resistant?MDR?K.pneumoniae represented by carbapenemase-producing and extended spectrum?-lactamase?ESBL?-producing strains are constantly appearing,especially the emergence of carbapenem-resistant hypervirulent strains in recent years has brought a more severe challenge to the treatment of K.pneumoniae infection.Bacteriophage has been recognized as an alternative or complementary therapy to antibiotics,which is considered as one of important solutions for MDR K.pneumoniae infection.However,like many other bacteria,phage resistance mutations can also occur during the cleavage of K.pneumoniae by phages,which greatly limit their therapeutic applications.In recent years,great achievements have been made in the studies of phage-host recognition and the mechanisms of phage resistance,but the related studies on K.pneumoniae were relatively lacking.Our previous studies also found that K.pneumoniae K7,a host strain of K2 serotype,was prone to produce phage-resistant mutant strains during the interaction with phage GH-K3,but the mechanism of its occurrence remains unclear.The clarification of the mechanism of phage resistance of K.pneumoniae is of great value in improving the therapeutic effect of phage on K.pneumoniae infection.Therefore,this study aims to reveal the mechanism by which phage GH-K3 induces phage resistance of K.pneumoniae K7.The biological characteristics of phage GH-K3,such as host spectrum and one-step growth curve,were firstly revealed in this study,and then the whole genome of the phage was sequenced and analyzed.These results showed that GH-K3 belonged to the Siphoviridae family with a burst size of 291 PFU/cell.The phage comprised of49,427 bp containing a total of 77 open reading frames?ORFs?and shared high degree of nucleotide similarity and close evolutionary relationships with 13 other phages.The above data revealed the biological characteristics of phage GH-K3,and laid a solid foundation for the subsequent study of phage resistance in host bacteria.The mechanism of phage resistance in K.pneumoniae was explored in the follow-up study.Our data showed that almost all the phage-resistant mutant strains derived from K.pneumoniae K7 under the pressure of GH-K3 had a rough-type colony morphology.A phage-adsorbing receptor mutation in the surface polysaccharides of a rough-type phage mutant strain represented by K.pneumoniae K7R^R was confirmed by centrifugation,scanning electron microscopy?SEM?,and phage adsorption assay,which was consistent with previous literature reports.In addition,it was unexpectedly found that a very small number of mutant strains had a smooth-type colony morphology,and named as K.pneumoniae K7R^B.The bacteriophage resistance of K7R^B was independent of surface polysaccharides,but seemed to be associated with the loss of protein-adsorbed receptors,which was found to be a novel phage resistance production pattern for K.pneumoniae.Bacterial surface polysaccharides had a leading role in the recruitment of GH-K3,but surface protein receptors appeared to be the decisive factor for phage infection.The above data revealed that the processes of K.pneumoniae resistance to phage infection were limited to the phage adsorption phase,further illustrating the possibility of K.pneumoniae having a“polysaccharide-protein”two-stage receptor adsorption mode.To elucidate the mechanism of phage resistance of K.pneumoniae to GH-K3,this study was conducted on K.pneumoniae K7R^R and K7R^B,respectively.The severe CPS synthesis defect in K7R^R has been confirmed by alcian blue staining.Additionally,it was verified by silver staining that the LPS components of the mutant strain did not change significantly,thereby determining that the primary phage adsorption receptor of GH-K3 was CPS instead of LPS.Compared to K7,K7R^R did not have a genetic mutation at the whole genome level.However,liquid chromatography-tandem mass spectrometry?LC-MS-MS?analysis showed that the abundance of three glucosyltransferases,GT-1,GT-2 and WcaJ,encoded by the gene cluster of Capsular Polysaccharide Synthesis?cps?was dramatically down-regulated.Further studies found that deletion of any of GT-1,GT-2 and wcaJ resulted in bacterial CPS synthesis defect accompanied by loss of GH-K3 sensitivity,indicating that all these three glucosyltransferases might be involved in maintenance of phage sensitivity.However,in the presence of macromolecular CPS residues?>250 KDa?,K7??GT-1?and K7??wcaJ?could still be bounded by GH-K3,though with a modest adsorption efficiency,suggesting that the CPS residues accumulated upon deletion of GT-1 or wcaJ did retain phage binding sites.Thus,the above data indicated that the phage resistance mutation of K7R^R and its rough-type colony morphology were associated with CPS synthesis defect caused by expression inhibition of three glucosyltransferases,GT-1,GT-2 and WcaJ.To reveal the phage resistance mechanism of K.pneumoniae K7R^B,differential genes and differentially expressed proteins between the mutant strain and K7 were analyzed in this study.In fact,ompC gene of K7R^B lacks one“G”between T711 and A712 compared with the wild type strain,which is the only genetic mutation site of the mutant strain.LC-MS-MS analysis showed that among differentially expressed proteins between between K7 and K7R^B,four outer membrane proteins in K7R^B,OmpC,OmpN,KPN₀2430 and OmpF,showed extremely significant expression inhibition.Gene complementation experiments showed that only K7R^B with ompC overexpression was cleaved by GH-K3.By contrast,deletion of ompC₇₁₂ in K7triggered OmpC silencing,resulting in resistance to GH-K3.These assays proved that OmpC was the key adsorption receptor for GH-K3,and the phage resistance of K7R^B was caused by mutation of its own ompC gene.In addition,the OmpC expression levels of three other K.pneumoniae strains with K2 serotype,KPP14,KPP27 and KPP36,were close to that of K7.The OmpC sequences of the four strains were identical except for residues 255,342,344 and 348,but the efficiency of plating of KPP27 by GH-K3 was very low,and KPP14 and KPP36 could not be infected by this phage.However,these strains became more sensitive to GH-K3 after their native ompC being replaced by ompC of K7,suggesting that OmpC^K7 was the most suitable secondary receptor for GH-K3.In summary,this study not only revealed phage resistance mechanism of K.pneumoniae K7R^R and K7R^B,but also demonstrated the two-stage receptor mode of“CPS-OmpC”during the adsorption of GH-K3 to host cells.Therefore,this study enriched the cognition of Klebsiella phage receptors,and it had certain reference significance for the subsequent studies of the phage resistance mechanisms of other bacteria.