Interactions Between The Bacterial Signaling Molecule CYCLIC-DI-GMP And Its Two Noval Receptors

Cyclic diguanylate monophosphate(c-di-GMP),a well-conserved second messenger in bacteria,participates in the regulation of many cellular processes,such as the biosynthesis of exopolysaccharide matrix substance in biofilms,bacterial motility,cell cycle progression,bacterial virulence and pathogen-host interactions.However,the c-di-GMP receptor has not been identified and the mechanisms by which c-di-GMP mediate pathogen-host interactions remain largely unknown in Mycobacterium tuberculosis,a human pathogen.In the present study,we identified two novel c-di-GMP receptors.One is human siderocalin(LCN2)involving in pathogen-host interactions,and the other is shikimate kinase(AroK)of Mycobacterium tuberculosis.Human siderocalin(LCN2)is a new c-di-GMP receptor.LCN2 belongs to the lipocalin family of immune proteins and can sequester bacterial siderophores to prevent iron acquisition and inhibit bacterial growth under iron-limited conditions.Bioinformatic screen characterized LCN2 as a putative receptor of c-di-GMP.Using surface plasmon resonance(SPR)and isothermal titration calorimetry(ITC)assays,we demonstrated that both c-di-GMP and ferric carboxymycobactins/ferric enterobactin(Fe-CMBs/Fe-Ent)could bind to rLCN2 protein,whereas rLCN2 could not interact with three c-di-GMP-like molecules(c-di-AMP,GTP and cGAMP)under the same condition.ITC assays showed that the binding stoichiometry between c-di-GMP against r LCN2 was 1:1 with the binding affinity value(Kd)1.63 ± 0.05 ?M,indicating that one molecule of c-di-GMP binds to one LCN2 monomer in the siderophore-binding pocket.Our results support a model in which,during infection,bacterial pathogens could utilize c-di-GMP as a weapon to hamper the interaction between siderophores and LCN2,and thus could inhibit the antibacterial activity of r LCN2.We identified shikimate kinase(AroK)as the first receptor of c-di-GMP in Mycobacterium tuberculosis.AroK is a key enzyme of shikimate pathway and cell wall metabolism,a potential target protein for anti-tuberculosis drug design.A protein expression library was constructed and it contains 80 proteins from Mycobacterium tuberculosis H37 Rv that have crystal structure information in Protein Data Bank.Using c-di-GMP binding screen,AroK was characterized as a candidate c-di-GMP receptor.Then,direct and specific interaction between AroK and 32P-labeled c-di-GMP was confirmed by a cross-linking assay.ITC experiments showed that the binding stoichiometry between c-di-GMP against AroK was 1:1 and the binding affinity value(Kd)is 2.453 ± 0.05 ?M,indicating that one molecule of c-di-GMP binds to one AroK monomer.Using purified mutant proteins,we found Ser16 located in the catalytic center of AroK is also key amino acid residue interacting with c-di-GMP.Interesting,c-di-GMP was shown to inhibit the enzymatic activity of AroK.Therefore,we identified AroK as the first c-di-GMP receptor in Mycobacterium tuberculosis.C-di-GMP directly binds to AroK and inhibits its catalytic activity.Our data suggest that AroK is involved in the regulation of mycobacterial cell wall metabolism,which may affect the growth and toxicity of pathogenic bacteria.Our findings provide a novel clue for shikimate kinase inhibitor design in Mycobacterium tuberculosis.