The Study Of Molecular Characteristics And Functions Of Iron Uptake Systems In Klebsiella Pneumoniae

Iron is involved in DNA replication,amino acid synthesis and electron transport for K.pneumoniae and thus it is essential for strains to survive and thrive.However,the low solubility naturally occurring oxidized forms of iron within host limits the iron bioavailability for K.pneumoniae.Besides,host iron is always bound with protein.For example,the majority of host iron is located within erythrocytes and carried by hemoglobin and iron in plasma is frequently bound by transferrin,while in other cells,iron is stored within ferritin.Therefore,K.pneumoniae strains have evolved versatile iron uptake systems(including siderophores,hemin uptake systems and iron ion uptake systems)to scavenge iron from host.The present study consists of three parts,covering the mechanisms of iron uptake systems contributing the virulence of K.pneumoniae.The first part mainly analyzed the epidemiological characteristics of acquired siderophore loci of K.pneumoniae in Zhejiang province and explored the association between mucoviscosity phenotype and siderophores.In this study,we isolated 272 K.pneumoniae strains from 20 tertiary hospitals in Zhejiang province and collected associated clinical information.All the strains were sequenced.In the 272 non-repetitive strains,123 fulfill the clinical definition of hv Kp and the rest 149 belonged to non-hv Kp.The analysis of cg MLST suggested that the genetic relationship among non-hv Kp was relative close while hv Kp strains were genetically diverse.We then focused on the analysis of the epidemiological characteristics of siderophores.The ybt loci for yersiniabactin were mainly located on ICEKp3(99/208,47.6%)and ICEKp10(69/208,33.2%).Ybt9 was predominant among MDR lineages ST11 and its single-locus variant ST4496 and was all located on ICEKp3;ybt1 was predominant in hypervirulent lineages ST23 and was located on ICEKp1 and ICEKp10.The prevalence rate of iuc and iro was 80.9%(220/272)and 55.5%(151/272)respectively,while 146(53.7%)of the strains simultaneously harbored iuc and iro.Among the iuc-/iro-positive strains,iuc1 and iro1 were predominant and were frequently located on p K2044-like virulence plasmids.In addition,with the increase of the siderophore types secreted by K.pneumoniae,the quantitive mucoviscosity also increased,indicating the association between mucoviscosity phenotype and siderophore production and the involvement of rmp A/rmp A2.The results of this part indicated that siderophore loci of K.pneumoniae were diversely distributed.Hypervirulent and MDR lineages had their predominant siderophore loci.Siderophore production reflected virulence levels and might synergistically interact with hypermucoviscosity phenotype.The second part analyzed the impact of iron uptake capacity of colicin I receptor(Cir A)on the virulence of K.pneumoniae.Based on the 272 K.pneumoniae genomes in the first part,we analyzed the distribution of cir A alleles.Cir A1 was the most frequent cir A alleles in hv Kp strains,while cir A198 was predominant in non-hv Kp.There were two amino acids differences between Cir A1 and Cir A198,locating the extracellular loops region and the side of β-barrel,respectively.Given that Cir A addtionaly transported catecholate siderophore enterobactin and salmochelin,we constructed fep A knockout mutant ZKP51 Δfep A and fep A and iro N knockout mutant D3 Δfep A Δiro N.Differences between Cir A1 and Cir A198 based on these two mutants were then analyzed.In irondepleted environment,Cir A1 transported iron-siderophore complexes more effectively and conferred higher serum resistance on K.pneumoniae,compared with Cir A198.In addition,Cir A is the critical gateway for the novel cephalosporin cefiderocol entering into bacterial cells.Mutation from cir A1 to cir A198 caused 4-fold increases in cefiderocol MICs,whereas the MIC of cefiderocol decreased 4-fold when cir A198 mutated to cir A1.These results suggested that the iron uptake ability of Cir A influenced the susceptibility to cefiderocol in K.pneumoniae.Furthermore,the transcriptome analyses indicated that the differences between Cir A1 and Cir A198 had impact on the expressions of ABC transporter and fimbria associated genes.The results of this part indicated that Cir A1 had stronger capacity in iron uptake than Cir A198,resulting in strains with Cir A1 possessed fitness under iron-depleted environment,higher serum resistance and susceptibility to cefiderocol.The third part analyzed the genetic diversity of hemin uptake system Hmu RSTUV of K.pneumoniae and verified its functions.K.pneumoniae strains sequester hemin and further utilize the iron carried by hemin via Hum RSTUV system.In this part,we collected 2242 K.pneumoniae genomes,of which,2218(98.9%)had complete hmu RSTUV locus.Based on the 2218 complete hmu RSTUV suqences,we established a novel type scheme named hm ST.Among the 2218 genomes,446 non-repetitive hm STs were identified.In hypervirulent lineages,hm ST was diversely distributed and hm ST1 mainly appeared in strains of ST23.In contrast,hm ST was less diversely distributed among MDR strains.Hm ST presented higher genetic diversity in hypervirulent lineages,bloodstream-sourced strains,clinical infection and community-acquired strains.Wild type and complemented strains outgrew strains of hmu R knockout in the M9 medium with bovine hemin supplement.However,there is no significant growth differences between wild and mutated strains in whole blood.Moreover,strains of hmu R knockout produced more siderophore productions,suggesting siderophores were feedback regulated by complete hmu system.In this part,we establish a novel typing scheme hm ST based on hmu RSTUV locus and further explored the association between epidemic hm STs and common K.pneumoniae lineages.Finally,we confirmed the biological function of Hmu R and its impact on the siderophore regulation.Overall,the present study uncovered the epidemiological characteristics of acquired siderophore loci of K.pneumoniae in Zhejiang province and researched the impact of nonspecific receptor of catecholate siderophore Cir A on the virulence and cefiderocl susceptibility.We also analyzed the genetic diversity of the hemin uptake system Hmu RSTUV.