Study On The Adhesion Between Bacterial Adhesin SfbⅠ And Fibronectin FnⅠ At Single Molecule Scale

Streptococcus pyogenes is an important human pathogen that causes common throat and skin infections such as pharyngitis and pustulosis,and it can also cause more serious invasive infections such as sepsis,toxic shock,rheumatic fever and acute nephritis.Current bacterial invasion of cells mainly involves the interaction between fibronectin binding proteins(FnBPs)located on the bacterial surface and α5β1 integrins on the host cell membrane.Fibronectin(Fn)is thought to mediate the bridge between FNBPs and integrins.Understanding how Fn interacts with bacterial FNBPs to mediate bacterial adhesion is of great biological significance,but the specific interaction mechanism is still unclear.In this study,single molecular force spectroscopy(SMFS)and molecular dynamics simulation(MDS)were used to reveal the molecular mechanism guiding the invasion of S.pyogenes cells,focusing on the interaction between the N-terminal domain 1-5F1 of fibronectin and the fibronectin binding protein SfbⅠ-5 of S.pyogenes.First,we constructed and expressed the related recombinant proteins of SfbⅠ-5 and 1-5F1,and then carried out atomic force microscopy(AFM)experiments by chemical modification.SMFS experiments showed that Sfb1-5 mediated the adhesion force of S.pyogenes to fiberectin 1-5F1 was about 1000pN,and this high affinity was mainly derived from the tandem β-zipper structure formed by the reverse parallel interaction between SfbⅠ-5 and 1-5F1.This structure has high mechanical stability,which makes FN a strong bridge for bacterial invasion and enhances bacterial adhesion to host cells.In addition,the results of molecular dynamics simulation provided us with the gradual dissociation process of 1-5F1-SfbⅠ-5 series β-zipper structure under external tensile force,which provided support for our experiment.The analysis of hydrogen bonding,tensile force and secondary structure between 1-5F1 protein and SfbⅠ-5 protein with time indicates that the high mechanical stability between 1-5F1 protein and SfbⅠ-5 protein is caused by the cooperation of multiple bonds.Our results provide a molecular basis for SfbⅠ to induce S.pyogenes to invade host cells,and provide a broad prospect for the development of therapies targeting intracellular pathogens.In addition,we speculate that AFM will help to identify compounds and effectively inhibit bacterial invasion of cells.