The Virulence Regulation Mechanism Of A Transcriptional Regulatory Factor MprA In Extraintestinal Pathogenic Escherichia Coli

Extraintestinal pathogenic Escherichia coli(ExPEC)is a significant zoonotic pathogen that causes severe infections in extraintestinal tissues,such as urinary tract infections and sepsis.Based on the evolutionary lineage,extraintestinal virulence genes,and infected sites,ExPEC is classified into urinary tract pathogenic E.coli,sepsis-related E.coli,and neonatal meningitis E.coli,among others.ExPEC is commonly grouped into B2 and D groups.The B2 group has the most diverse gene pool among E.coli lineages,encoding virulence factors such as adhesins,iron acquisition systems,proteases,and toxins.ExPEC causes systemic infections,including bacteraemia,after initial infection and can lead to death in severe cases,causing significant losses to human health and the global livestock industry.MprA is a member of the MarR transcriptional regulatory factor family.MprA binds DNA to prevent RNA polymerase from binding to the promoter,thereby suppressing target gene transcription.It can also regulate the expression of downstream genes in response to environmental changes.Upon binding small molecule ligands such as naphthoquinones,salicylic acids,and DUO11,MprA’s DNA binding ability decreases,thereby losing its regulatory function on target genes.Recent studies have shown that MprA not only regulates the classical function of multidrug efflux pump Emr AB,but also affects the expression of E.coli capsular genes and the virulence genes of Salmonella SPI-2.Our laboratory screened essential genes related to sepsis in mammalian and avian models of ExPEC XM and found that MprA is critical for the virulence of ExPEC XM.However,there is currently no literature reporting which genes are affected by MprA in the ExPEC genome,so analyzing the genes regulated by MprA in ExPEC and its regulation mechanism is of great significance.In this study,we used a combination of transcriptome sequencing(RNA-Seq)and chromatin immunoprecipitation sequencing(ChIP-Seq)to clarify the target genes of MprA within the ExPEC XM genome.Then,we verified the impact of MprA on the transcription level and function of target genes through real-time fluorescence quantitative PCR and in vitro experiments.We explored the effect of MprA and target genes on the virulence of ExPEC XM in a mouse model.Finally,we clarified the regulatory mechanism of MprA and the mechanism by which it exerts virulence.First,to clarify which genes MprA regulates within the genome,we combined previous RNA-Seq data from our laboratory with ChIP-Seq data obtained in this study and found that MprA directly regulates99 genes and indirectly regulates 667 genes in ExPEC XM.At least 13.44% of genes in the ExPEC XM genome were affected by MprA,revealing it as a global transcriptional regulator.Gene Ontology(GO)analysis of the differential genes showed that MprA primarily affected genes related to metabolism,adaptation,transport,cell structure,and regulation in ExPEC XM.In the verification experiment of the capsular gene cluster,we observed that MprA can activate the expression of the capsular gene cluster.ChIP-Seq confirmed that MprA can bind to the promoter region of the initial gene kps F of the capsular gene cluster,proving that MprA directly regulates the expression of the capsular gene cluster.The K1 capsular is related to resistance to host macrophages phagocytosis and serum killing.In the anti-phagocytosis experiment,the phagocytosis rate was growth for ExPEC XM,Δneu,ΔmprA,ΔmprAΔneu,respectively.The complemented strain ΔmprA-C partially restored the phagocytic resistance of the strain.This proves that MprA plays an important role in resisting phagocytosis by host macrophages in ExPEC XM.We also found that the survival ability of the strain in serum decreased significantly after deletion of mprA and neu,and the survival ability in serum was partially restored by the mprA complement strain.This indicates that MprA can resist serum killing by regulating the expression of the K1 capsule.Our animal experiments evaluating the pathogenicity of the deletion strain showed that the deletion of the neu and mprA genes significantly reduced the bacterial load and virulence in the blood of the mouse model,proving that MprA exerts virulence by regulating the expression of the capsular gene cluster.The above results indicate that MprA directly regulates the expression of the K1 capsule gene cluster,resists phagocytosis by host macrophages,resists serum killing,and exerts virulence.The verification experiment on the acid sensing signal system gene evgAS showed that MprA can activate the expression of evgAS.ChIP-Seq analysis found that MprA can bind to the promoter region of evgA,proving that MprA directly regulates the acid sensing signal system gene evgAS.Results from the in vitro acid tolerance test showed that the survival rate of ΔmprA and ΔevgS was significantly decreased,while the complemented strains could restore the survival ability under pH 2.5 conditions.Transcriptome data also showed that MprA can also affect the expression of acid-related genes hde BAD and gadE.Therefore,MprA directly activates the acid sensing signal system gene evgAS and acid-related genes hde BAD and gadE to exhibit acid tolerance in vitro;indicating that MprA plays an important role in resisting acid stress in ExPEC XM.Animal experiments showed that the deletion of evgS had little effect on the virulence in the mouse infection model,indicating that the acid sensing signal system Evg AS may play a role in other physiological processes of ExPEC-host interaction rather than being essential in the process of ExPEC entering the bloodstream.In the functional verification experiment of the T2SS gene cluster,it was observed that MprA can activate the expression of the T2SS gene cluster.ChIP-Seq analysis found that MprA has specific binding peaks in the promoter region of the first gene sslE in the T2SS gene cluster,so MprA directly activates the expression of T2SS gene cluster.Through secretome analysis and protein mass spectrometry,it was found that MprA can affect the expression of SslE and regulate the secretion of SslE through the regulation of GspD,which is related to T2SS secretion function,thereby affecting the formation of the ExPEC XM biofilm.Animal experiments showed that the deletion of GspD and SslE in T2SS had little effect on the virulence in the mouse infection model,indicating that MprA can directly activate the expression of T2SS and participate in virulence to a certain extent.In the functional validation experiment of the lactose operon,it was observed that MprA can bind to lac I internally and directly activate the expression of the lactose operon genes,increasing the activity units of beta-galactosidase LacZ.In the indirect regulation target gene validation experiment,it was observed that MprA can inhibit the expression of I-F CRISPR in ExPEC.We found that salicylic acid and naphthoic acid can form ligand complexes with MprA in the wild-type strain,silencing its DNA-binding function,thereby relieving the inhibitory effect of MprA on emr AB and I-F CRISPR genes.This also indirectly confirms MprA’s regulatory role in the I-F CRISPR system.However,no binding site of MprA protein was found in the IF CRISPR promoter region in the ChIP-Seq results,indicating that the I-F CRISPR gene cluster is regulated indirectly by MprA.We also observed that the expression of I-E CRISPR in K12 was downregulated after the loss of mprA,suggesting that MprA may also be an activator of the I-E CRISPR gene cluster.MprA is encoded in the core genome of E.coli.To explore whether MprA has the same regulatory effect on specific genes in other ExPEC and non-pathogenic E.coli,we respectively detected the effect of MprA on target genes in the representative strains of ExPEC RS218,UTI89,CFT073,and nonpathogenic E.coli K12.It was found that MprA has a universality in the regulation of common target genes of E.coli.It can inhibit the expression of type 1 pilus and I-F CRISPR system genes,and activate the expression of acid-sensing signal system genes evgAS,Group 2 K1 and K2 capsule-related genes.In summary,this research systematically explored the regulatory network and regulatory mechanism of MprA in ExPEC,clarifying the pathogenesis of MprA as a regulator protein and determining MprA as a potential drug target,providing a theoretical basis for the development of anti-MprA antibiotics.