Molecular Mechanism Of Iron Metabolism Regulation By YdiV And SlyD In Escherichia Coli

The transition metal iron is essential for bacterial survival and growth.As an indispensable cofactor,iron is involved in many cellular processes,including N2 fixation,DNA synthesis,and respiration.However,iron limitation is a challenge for bacteria due to the extremely low solubility of the Fe3+ ion.For pathogenic bacteria,this challenge is even more severe since humans and other mammals have evolved a complicated mechanism of nutritional immunity to restrict iron availability.As a result,the concentration of free iron in the plasma of humans is much lower than that required for bacterial growth.Consequently,pathogenic bacteria must overcome the nutritional immunity to successfully establish infection.Escherichia coli have evolved many strategies to acquire sufficient iron from their environment or host.Synthesis and secretion of siderophore,such as enterobactin(Ent),is a dominant strategy used by E.coli to salvage iron.Ferric-Ent is recognized by FepA,an outer membrane receptor,and transported into the periplasm in a TonB-dependent process.Once in the periplasm,ferric-Ent is immediately captured by FepB and then delivered by the ATP-binding cassette(ABC)transporter,FepDGC,into the cytoplasm where the Fe-Ent can be utilized.Intriguingly,iron is not only necessary,but it is also dangerous for bacteria.A high intracellular Fe(?)concentration can trigger a Fenton reaction,causing cell toxicity.Therefore,bacteria must keep intracellular iron levels at appropriate concentrations in order to satisfy their physiological needs while avoiding harm.Regulation of the intracellular iron homeostasis is dependent upon the ferric uptake regulator(Fur)protein.Fur is a dimeric protein that is highly conserved across many bacteria.The Fur monomer binds a structural Zn2+ and has a regulatory binding site for the Fe2+.The mechanism of Fur in ferric regulation has been well established.Under iron-sufficient conditions,Fe2+ binds to the regulatory site,and the Fur homodimer then combines with the operator site of a target promoter,blocking the binding of the RNA polymerase holoenzyme(RNAP)and inhibiting iron-uptake genes expression.While under iron-limited conditions,Fur releases the regulatory Fe2+ and dissociates from the target DNA sequence,thus relieving the iron-uptake gene repression.This classic model relies on the feedback regulation of Fe2+-Fur binding.In recent years,however,scientists have shown some diverse results in the regulation of homologous Fur proteins.For instance,both the ion-bonding site 1 and site 2 mutants of Bradyrhizobium japonicum Fur were able to repress gene expression in vivo.The Campylobacter jejuni apo-Fur is able to dimerize and bind to its target promoter DNA sequence.These phenomena imply that there are other mechanisms regulating Fur protein functionFlagella also play important roles in the invasion of pathogenic bacteria,but it is also a strong signal of danger to the host immune system.In order to successfully invade and avoid recognition by the host's innate immune system,pathogenic bacteria must precisely control their flagella expression levels during adhesion,invasion,and colonization.As such,it no surprise that recent studies have found that the regulation of iron homeostasis and flagellar biogenesis are coordinated in some bacteria.However,the underlying mechanism is unknownIn E.coli,the expression of flagella is tightly controlled by the master regulator FlhD4C2 complex.The transcriptional function of FlhD4C2 is inhibited by YdiV,a degenerate EAL domain protein,in a concentration-dependent manner.This negatively regulates flagella biogenesis and,thus,bacterial motility.Interestingly,YdiV is induced under nutrient-starvation conditions,and its homologue is highly upregulated during invasion of Salmonella,which suggests its important role in bacterial infectionIn this study,we found that YdiV,the flagella gene inhibitor,is involved in iron homeostasis in E.coli.The main research contents and conclusions of this paper are as follows:To test the association of ydiV with iron homeostasis,we used qRT-PCR to test the expression of ydiV or iron uptake system genes(fepA,fecB,and fhuF)in wild-type E.coli MG 1655,?ydiV,and ?ydiV-pTracydiV strains.As the results,in iron-deficient environment,the expression of ydiV is up-regulated.High concentrations of YdiV can promote the transcription of iron absorption system genes increased significantly.The ICP-MS results shown that,the content of iron in E.coli is positively correlated with the content of YdiV.We used ?-galactosidase enzyme activity assays to detect the transcriptional activity of fepA and fhuF gene promoters in strains with different YdiV content,and found that high concentrations of YdiV in cells can promote Fur's de-inhibition of iron metabolism gene promoters.If YdiV and Fur are simultaneously overexpressed in E.coli BL21(DE3),the YdiV-Fur binary complex cannot be obtained,and only Fur is purified(FurYdiV).However,the results of EMSA and FP experiments showed that the affinity of purified FurYdiV to Fur box DNA was about 300 times lower than that of wild-type Fur.Therefore,we believe that YdiV interacts with Fur in the cell,and the effects of this interaction can exist stably,and will not be restored by the dissociation of YdiV.In order to explore the molecular mechanism by which YdiV affects the binding of Fur to DNA,we conducted a multi-faceted detection and comparison of Fur and FurYdiV,which have significant differences in affinity with Fur box DNA.The results of size-exclusion chromatography,ICP-MS and ITC showed that the polymerization state and metal ion binding properties of the two proteins were not significantly different.However,many clues suggest that the structures of them are different.Firstly,in size-exclusion chromatography,the dimer Fur always lags behind the dimer FurYdiV slightly,which shows that the shape of Fur and FurYdiV is different.Secondly,through high-resolution mass spectrometry and sulfhydryl detection experiments,it was confirmed that Fur contains a pair of disulfide bonds,while FurYdiV does not.In other words,the increase of intracellular YdiV causes the disulfide bond of Fur to be reduced Furthermore,VP-DSC experiments show that FurYdiV has higher thermal stability than Fur,that is,YdiV makes Fur's conformation more stableSince YdiV does not have isomerase or disulfide bond reductase activity,we speculate that there are other unknown proteins involved in the interaction between YdiV and Fur.Fortunately,we used pull-down experiments combined with mass spectrometry sequencing technology to find this key protein SlyD.SlyD is a prolyl cis/trans isomerase and molecular chaperone.The EMSA experiment showed that in AslyD strain,overexpression of YdiV could not inhibit Fur binding with DNA.The DBD domain of E.coli Fur contains two key proline residues:P18 and P29.Using site-directed mutagenesis to perform amino acid mutations on Fur,it is found that the DNA binding activity of Fur PI 8A is no longer regulated by YdiV and SlyD,that is,the action site of SlyD on Fur is P18.Bacterial two-hybrid experiments proved that there are intracellular interactions among YdiV,Fur and SlyD.NMR experiments confirmed that wild-type Fur and FurYdiV,SlyD have obvious conformational differencesIn order to explore the physiological significance of the YdiV-Fur-SlyD regulation model,we first used in vitro transcription experiments to compare the inhibition efficiency of the classic Fur regulation model and the YdiV-Fur-SlyD regulation model on the fepA gene promoter.Results shown that the new YdiV-Fur-SlyD regulation model can activate the transcription of iron uptake genes more thoroughly.Mobility experiments shown that the iron-regulating proteins SlyD and Fur will not affect the flagella regulation function of YdiV,which is beneficial to the coordinated regulation of flagella synthesis and iron metabolism when pathogenic microorganisms invade host cells.Finally,bacterial invasion experiments were used to demonstrate the important function of YdiV and SlyD for UPEC pathogenicityThrough the above results,we found that the expression of the ydiV gene was upregulated in an iron-deficient environment,and high levels of YdiV transforms Fur into a novel form,which does not bind DNA in a SlyD-dependent manner.This form of Fur(FurYdiV,SlyD)contains the same number of Zn2+ and Fe2+ ions as the active native Fur,but it has different conformation.Thus,at least in E.coli,Fur can sense the concentration of YdiV to maintain iron homeostasis,and the expression level of YdiV is associated with the intracellular iron concentration.Thus,our findings established a new model about how E.coli employs the Fur-YdiV-SlyD axis to maintain iron homeostasis under different iron conditionsIn an iron-rich environment,the expression of YdiV is low and there is certain amount of SlyD inside the bacterial cell.Transcription and translation of fur gene are maintained at a steady rate.Most of the nascent Fur peptides fold into a dimer with the normal conformation containing a disulfide bond in each monomer(Folding pathway I,native Fur).Some of the remaining nascent peptides are recognized by SlyD,but without the help of YdiV,only a small fraction of them fold into the new conformation Since normal Fur is dominant under this condition,most Fur box DNA is occupied by Fur,which occludes the RNA polymerase and represses the corresponding iron acquisition genes.When E.coli enters an iron-deficient environment,such as invasion of host cells,the expression of YdiV is stimulated by unknown mechanisms.A high concentration of YdiV promotes the formation of a transient tertiary YdiV-Fur-SlyD complex,which reduces the disulfide bonds in Fur and leads to a more stable Fur without DNA-binding ability(Folding pathway ?,FurYdiV,SlyD).Once FurYdiV,SlyD is produced,the transient tertiary complex dissociates releasing YdiV and SlyD to catalyze formation of more FurYdiV,SlyD.Consequently FurYdiV,SlyD becomes dominant and most of the Fur box DNA is free,which de-represses iron acquisition genesThe mechanism described above depends on the peptidyl-prolyl cis-trans isomerase activity of SlyD,which targets Pro18 of Fur.During this process,YdiV functions as an essential helper molecule of SlyD making isomerization reactions easier and faster.Nutritional immunity and pattern-recognition receptors(PRRs),triggered following immune reactions,pose deadly threats to intracellular pathogens.Strikingly,flagellin can be detected by the host PRR TLR5,which triggers pyroptosis of macrophages.To survive and grow inside the host cells,intracellular pathogens have developed complex signaling systems to counteract host immunity.Formation of the YdiV-Fur-SlyD axis is an effective mechanism to tightly coordinate the countermeasures of the bacteria,which enables UPEC to grow inside and outside of the host cells.The bacterial invasion experiments also proved the important influence of YdiV and SlyD on the pathogenicity of UPEC.These results have important guiding significance for the development of new antibiotics targeting iron uptake pathways.