Screening For In Vivo Fitness Factors Of Procine Extraintestinal Pathogenic Escherichia Coli By Transposon Insertion Sequencing Technology

Extraintestinal pathogenic Escherichia coli(ExPEC)is an important human-animal pathogen that causes urinary tract infection,meningitis and septicemia in humans,and can cause extra-enteric infection and even death in poultry and cattle in animals.In recent years,ExPEC has gradually become prevalent in swine herds,and the infection leads to serious diseases such as meningitis,pneumonia and septicemia in pigs,causing serious harm to the pig industry.Currently,there is no vaccine for ExPEC in pigs,and traditional antibiotic treatment is often ineffective due to drug resistance problems,and effective control strategies and tools need to be developed.Pathogenic bacteria use specific virulence factors or in vivo fitness factors to promote their survival and pathogenicity in vivo,therefore,deeper excavation of pathogenicity-related factors of pathogenic bacteria and revealing the role of these factors in the infection process are essential to understand the pathogenic mechanism of pathogenic bacteria,and have important theoretical significance for the discovery of novel antibacterial drugs and vaccine targets.At present,our understanding of porcine ExPEC is not deep enough and its pathogenic mechanism is not sufficiently studied.Traditional virulence factor identification methods are inefficient,laborious,and difficult to discover new virulence factors.Therefore,in this study,we constructed a porcine ExPEC transposon mutant library using the random insertion property of transposons,identified genome-wide in vivo fitness factors and serum survival factors of porcine ExPEC using a mouse model and transposon insertion sequencing technology(TraDIS),and validated against important genes,and obtained the following main findings:1.A Mariner transposase-based random transposon mutant system was constructed and successfully implemented in the clinical isolate of the strong virulent strain ExPEC PCN033 using conjugation transfer.Using this transposon mutagenesis system,a library of transposon mutants of ExPEC PCN033 strain was constructed,which contains about68,000 monoclonal transposon mutants.High-throughput sequencing of this library revealed that the number of genes containing transposon insertions in this library accounted for 72% of the genes encoded in the entire genome.Thus,a near-saturated ExPEC PCN033 random mutant library was successfully constructed in this study.2.Using this ExPEC transposon mutant library(Input)to infect mice,bacteria(Output)were isolated from brain,spleen and lung tissues after 12 h.The libraries were sequenced in high throughput to compare the transposon insertion frequencies of each gene in the Input and Output libraries,and a total of 100 differential genes were identified in brain tissue,83 differential genes in spleen tissue and 92 differential genes were identified in lung tissue.Among them,64 ExPEC differential genes were identified in both and more tissues.KEGG enrichment analysis revealed that 42 genes among the screened genes were located in metabolic pathways,including 6 genes in membrane transport pathways,and COG functional clustering analysis revealed a high proportion of genes related to ion channels and cell wall structures,containing 11 and 5 genes,respectively.3.The above screened ExPEC PCN033 in vivo adaptability factors were further validated by constructing a total of 23 gene deletion mutants,and experiments revealed that the rfa manipulator deletion may affect the ability of ExPEC to colonize mice by affecting the survival of bacteria in serum and thus the ability of ExPEC to colonize mice;ΔfimG significantly reduced the adhesion ability of ExPEC to epithelial cells and its colonization ability in mice,and is an important in vivo fitness factor of ExPEC.Deletion of metabolism-related genes,such as fepB,sdhC and malM,could affect the colonization ability of ExPEC in mice by affecting different metabolic functions.Deletion of regulation related genes baeS and metJ reduced the colonization ability of ExPEC in mice,adhesion to epithelial cells and resistance to macrophage phagocytosis,which are important factors involved in the pathogenesis of ExPEC.4.The above-constructed ExPEC PCN033 transposon mutant library was used to screen genes with different transposon insertion frequencies before and after serum treatment,and 32 genes associated with serum survival were screened.A single plasmid CRISPRi gene expression silencing system in ExPEC PCN033 strain was constructed and validated to find xylF,torC,PPECC33＿RS04820 and other genes associated with ExPEC PCN033 serum survival.In this study,we successfully constructed a transposon mutant library of ExPEC PCN033 strain,and explored the genome-wide genes of ExPEC in vivo fitness factors and serum survival-related genes using TraDIS technology,and explored the role of related genes in the pathogenesis of ExPEC,which laid the foundation for the in-depth understanding of the pathogenesis of ExPEC and the exploration of novel antibacterial drug targets.