Identification Of Virulence Genes In Extraintestinal Pathogenic Escharichia Coli And Virulence Effect Of MprA And NhaA

Extraintestinal pathogenic Escherichia coli(ExPEC)is an important zoonotic pathogen that can cause severe infection of extraintestinal tissues in different hosts such as humans and animals,mainly including Urinary pathogenic E.coli(UPEC),Meningitis/Sepsis associated E.coli(MNEC),and Avian pathogenic E.coli(APEC).UPEC is the most common pathogen of urinary tract infections,which can cause cystitis and pyelonephritis;MNEC can cause sepsis and is the main cause of bacterial meningitis in newborns;APEC is one of the three most important infectious pathogens of poultry,mainly causing balloon inflammation,pericarditis,perihepatitis,and sepsis.In recent years,with the increase of the antibiotic resistance of ExPEC,more and more people and animals are infected with pathogenic E.coli,which is difficult to treat,leading to an increase in mortality,which seriously harms human and animal health,and causes huge economic losses to the aquaculture industry.So far,many virulence factors of ExPEC have been determined to play an indispensable role in the infection process,but there are still some virulence factors that have not been found and the pathogenic mechanism is unclear.Furthermore,ExPEC also has zoonotic potential.In addition to causing human infection,it can also infect many farm animals,companion animals,and wild animals,especially poultry,causing huge economic losses to the poultry industry.This means that host-specific virulence factors may be present.Therefore,the discovery and study of ExPEC virulence genes,host-specific virulence factors and pathogenic mechanisms are prerequisites for the treatment and prevention of ExPEC infection.With the continuous development of sequencing technology,the identification of virulence genes and the study of pathogenic mechanisms by researchers have exploded.More and more studies focus on the direct relationship between genotype and phenotype,genome sequencing analysis,transcriptome sequencing analysis,proteome sequencing analysis,and chromatin immunoprecipitation analysis have become the most commonly tools to study the entire genome of the bacteria.In addition,due to its huge advantages in studying the relationship between bacterial genotypes and phenotypes,transposon insertion sequencing has gradually become a common method for identifying virulence genes and pathogenic mechanisms.To identify new virulence factors,host-specific virulence factors,and explore their pathogenic mechanisms in ExPEC,this study first used transposon insertion sequencing technology to identify virulence factors in mammalian and avian sepsis models,and compared host-specific virulence factors between mammals and birds.Next,the two genes,mprA and nhaA,were selected to explore the pathogenic mechanism.Using transcriptome sequencing analysis,the differentially expressed genes of wild strain and mprA-mutant strain were compared,and their pathogenicity and how mprA regulated these genes were studied.At the same time,using site-directed mutation technology,the contribution of different nhaA loci to the virulence of ExPEC was investigated.1 Identification of virulence genes in extraintestinal pathogenic E.coli in different hostsExtraintestinal pathogenic Escherichia coli(ExPEC)is an important human and animal pathogen.Despite the apparent similarities in their known virulence attributes,some ExPEC strains can cross the host species barrier and present a zoonotic potential,whereas other strains exhibit host specificity,suggesting the existence of unknown mechanisms that remain to be identified.We applied a transposon-directed insertion site sequencing(TraDIS)strategy to investigate the ExPEC XM strain,which is capable of crossing the host species barrier,and to screen for virulence-essential genes in both mammalian(mouse)and avian(duck)models of E.coli-related septicemia.We identified 151 genes essential for systemic infection in both mammalian and avian models,97 required only in the mammalian model,and 280 required only in the avian model.Ten genes/gene clusters were selected for further validation,and their contributions to ExPEC virulence in both mammalian and avian models or mammalian-or avian-only models were confirmed by animal tests.The results showed that the neu-kps,rml,mprA,nhaA,yga,and sanA genes/gene clusters played important roles in both mammalian and avian models of ExPEC infection,while the bio,ere,and rnf gene clusters were only critical for ExPEC infected avian model,Type VI secretion system genes are only important for mammalian models.These genes may be ideal targets for the development of widely conserved vaccine antigens and for the development of new antibacterial drugs.This represents the first comprehensive genome-wide analysis of virulence-essential genes required for systemic infections in two different host species and provides a further comprehensive understanding of ExPECrelated virulence,host specificity,and adaptation.2 RNA-seq reveals regulatory network of MprA in extraintestinal pathogenic E.coliFor a long time,the transcriptional regulator MprA has been considered as an important repressor of the multidrug-resistant pump EmrAB which is involved in the regulation of E.coli antibiotic resistance.In recent years,studies have shown that MprA is closely related to capsule synthesis and can promote the expression of multiple capsule genes in E.coli.In previous studies,the results of TraDIS and animal experiments confirmed that mprA is an important virulence gene for ExPEC.However,the entire regulatory network of MprA is still unknown,and there may be many genes essential for infection.In this study,we used transcriptome sequencing analysis(RNA-seq)and Electrophoretic Mobility Shift Assay(EMSA)to explore the regulatory network of MprA.RNA-seq results showed that compared with ExPEC XM,152 genes were differentially expressed in ?mprA,and multiple gene clusters were up-regulated or down-regulated.Real-time PCR results show that the expression of differential genes is highly consistent with the results of RNA-seq,proving the reliability of RNA-seq results,but RNA-seq cannot distinguish whether MprA regulates these genes directly or indirectly.Therefore,we selected the most significant differential genes(capsule-related genes,emrAB,and CRISPR-related genes)to predict the promoter regions of these genes and interact with the expressed and purified MprA.EMSA results confirmed that MprA can directly regulate capsular-related genes,emrAB and CRISPR-related genes,which means that MprA can affect virulence,drug resistance and resistance to foreign genes.This study explored the regulatory network of MprA and confirmed its direct regulatory effect on some genes,which deepened our understanding of the regulatory network and pathogenesis of ExPEC.3 Effects of different NhaA sites on the virulence of ExPECNa+/H+antiporter is a ubiquitous membrane protein and NhaA is the most typical in ExPEC.NhaA has 12 transmembrane regions and plays an important role in the ionic homeostasis of cells.In previous studies,we found that the main Na+/H+antiporter NhaA of parenteral pathogenic E.coli plays an important role in virulence,and ?nhaA has a significantly reduced virulence in mammalian and avian models.In this study,we verified the role of ?nhaA in antiphagocytosis and serum survival,and found that NhaA has nothing to do with the survival of ExPEC in macrophage Raw264.7,but it plays an important role in human serum survival.At the same time,site-directed mutagenesis and animal experiments were used to verify the effects of different NhaA sites on the virulence of ExPEC.It was found that the sites of mutations affecting Na+and Li+transport rates were not related to the virulence of ExPEC.Na+and Li+transport rates and pH-sensing sites have less effect on the virulence of ExPEC,and the virulence of NhaA protein inactivation sites is significantly reduced in animal models,and in addition to Loop 1 and Loop 7 of NhaA,other Loop is also critical for the virulence of ExPEC.This evidence proves that the intact NhaA antiporter is essential for the survival of parenteral pathogenic E.coli in the blood of infected animals,so the sites or loops that affect the integrity of NhaA are promising for the preparation of unconventional drugs and new target of the vaccine.