Mechanisms Of Extraintestinal Pathogenic Escherichia Coli RS218 Against Iron-restricted Environments And Host Complement Attack

Extraintestinal Pathogenic Escherichia coli(ExPEC)is responsible for infections in both humans and farm animals(e.g.,urinary tract infections,bacteremia,neonatal meningitis,and sepsis),which impose a substantial burden on both public health and economics.In contrast to commensal E.coli,ExPEC encodes a series of virulence factors that enable it to survive and establish infection in hostile,nutrient-deficient hosts.The host’s innate immune system is critical in identifying and eliminating invading microorganisms.The low iron environment in the host limits the uptake of iron,an essential nutrient,by pathogens,further hindering the colonization and survival of pathogens in the host.The complement system is a core component of innate immunity and is considered the first line of defense against pathogenic microorganisms.The complement system can not only directly eliminate pathogenic bacteria,but also promote the phagocytosis of pathogens by macrophages through opsonization.ExPEC includes multiple pathogenic types,such as uropathogenic E.coli(UPEC),neonatal meningitis E.coli(NMEC),septicemic E.coli,poultry pathogenic E.coli,and avian pathogenic E.coli(APEC).Different pathogenic types of ExPEC are highly similar in serotypes,virulence factors,and pathogenic mechanisms.ExPEC can avoid the host’s nutrient restriction and complement attack,leading to systemic infection of the host.Exploring the mechanism by which ExPEC escapes iron-restricted environment and complement attack would broaden further research into the molecular mechanism of ExPEC pathogenicity and provide a theoretical basis for the prevention and control of E.coli.1.Effects of ExPEC binding to transferrin and utilizing iron on its growth and survivalTransferrin(TF)is an important Fe-storage protein in the host.Some pathogenic bacteria can acquire iron by binding to TF.It was found that the ExPEC strain RS218 could utilize iron in iron saturated holo-TF for its growth and survival in limiting medium,heat-inactivated serum,and macrophages.Through colloidal gold transmission electron microscopy,fluorescence microscopy,and fluorescence intensity analysis,it was found that ExPEC strain RS218 could bind holo-TF on the bacterial surface,but not iron-free apo-TF.Fluorescence detection also found that ExPEC in macrophage THP-1 could also bind to intracellular holoTF.In this study,through calcein fluorescence quenching and holo-TF conversion assays,it was confirmed that ExPEC could take up iron in holo-TF.Western blotting did not detect TF in the cytoplasm of ExPEC strain RS218,indicating that ExPEC does not take up holo-TF into the cytoplasm.The above results indicate that ExPEC strain RS218 binds holo-TF on the bacterial surface and takes up iron in it,which establishes a foundation for the study of holo-TF-binding proteins on the surface of ExPEC.2.Mechanism of EFG binding to holo-TFIn this study,desthiobiotin pull-down,two-dimensional electrophoresis,and mass spectrometry were used to screen and identify holo-TF receptors on the surface of ExPEC.It was found that elongation factor G(EFG)is a potential TF-binding protein.The interaction between EFG and holo-TF was confirmed by Far-Western blotting,ELISA plate binding assay,desthiobiotin pull-down,and protein inhibition assay.EFG was able to specifically bind holo-TF but not apo-TF.By detecting the production of apo-TF after the interaction between holo-TF and EFG,it was found that EFG can release iron in it.By constructing recombinant proteins with deletion of EFG domains or fragments,it was found that the Nterminal domains of EFG has the ability to bind holo-TF,and the C-terminal domains has the function of releasing iron in holo-TF.Using the exogenous protein surface expression vector,the EFG and its domains were expressed on the surface of non-pathogenic E.coli strain BL21.The binding of EFG and its domains to holo-TF and the release of holo-TF were demonstrated at the whole bacterial level.Iron two functions.Overexpression of EFG on the bacterial surface can significantly increase the binding of holo-TF of bacteria in macrophages,and significantly improve the survival ability of bacteria in macrophages.These results reveal a novel iron acquisition mechanism involving EFG,which suggests novel research avenues into the molecular mechanism of ExPEC resistance to nutritional immunity.3.The mechanism of EFTu binding to holo-TFElongation factor Tu(EFTu)of ExPEC strain RS218 was also screened as a potential holo-TF binding protein.In this study,EFTu was identified as the surface protein of ExPEC by immunofluorescence,colony blotting,and Western blotting.The specific binding of EFTu to holo-TF was confirmed by Far-Western blotting,ELISA plate binding assay,and protein inhibition assay.EFTu was not bound to apo-TF by ELISA plate binding assay.By detecting the production of apo-TF after the interaction between holo-TF and EFTu,it was found that EFTu can release iron from holo-TF.The EFTu domain-deleted proteins were constructed,and each domain of EFTu was found to be involved in binding holo-TF and releasing iron in holo-TF.Using surface overexpression vectors,it was found that overexpression of EFTu in non-pathogenic E.coli not only enhanced the ability of bacteria to bind holo-TF,but also promoted the acquisition of iron in holo-TF.Serum survival assays showed that EFTu significantly improved the survival ability of E.coli in heat-inactivated human serum,and this promotion was positively correlated with holo-TF,but not with apo-TF.Our research revealed a novel function of EFTu in binding holo-TF to promote iron uptake by bacteria,suggesting that EFTu was a potential virulence factor of ExPEC.In addition,our study provided research avenues into the iron acquisition and pathogenicity mechanisms of ExPEC.4.Mechanism of ExPEC binding factor H to escape complement attackFactor H(FH)is an important regulatory protein of the complement system.In this study,ExPEC strain RS218 was found to bind FH from serum.A method that combined desthiobiotin pull-down and liquid chromatography-tandem mass spectrometry was used to screen the FH-binding membrane proteins of ExPEC.Screening results revealed multiple carbohydrate metabolic enzymes(CMEs),including acetate kinase,fructose-bisphosphate aldolase,fumarate reductase flavoprotein subunit,L-lactate dehydrogenase,dihydrolipoamide dehydrogenase,phosphoenolpyruvate,synthase,and pyruvate dehydrogenase,were FH-binding proteins.The GST pull-down assays were found that these seven proteins can interact with FH from serum.The ELISA plate binding assays confirmed that seven proteins bind FH in a dose-dependent manner.The higher the protein concentration,the stronger the ability to bind FH.The protein inhibition assays demonstrated that each protein could significantly reduce the recruitment of ExPEC to FH(P<0.05).The above experiments revealed that these seven CMEs were able to recruit FH from serum.The immunofluorescence,colony blotting,and Western blotting targeting outer membrane proteins determined that these seven CMEs were located on the outer membrane of ExPEC strain RS218.The FH recruitment levels and C3b deposition levels on bacteria were significantly increased and decreased in an FH-concentration dependent manner,respectively by ELISA.The opsonophagocytosis assay showed that FH recruitment significantly enhanced the ability of ExPEC to resist opsonophagocytosis of human macrophage THP-1(P<0.05),and the more FH recruited,the stronger the anti-opsonophagocytic ability of ExPEC.This study revealed a novel mechanism by which ExPEC utilizes CMEs on the bacterial surface to bind FH and reduce the deposition of C3b on the bacterial surface,thereby enhancing the anti-opsonophagocytic ability to evade complement killing.