Structural Characterization And Mechanistic Investigation Of The Flagellar Assembly Protein FlgL-FliD Complex Of Salmonella Typhimurium

Bacterial movement mainly relies on the rotation and propulsion of flagella.By adjusting the number and swing speed of flagella,bacteria can quickly swim in a liquid environment,and can also adhere to host cells through flagella,enhancing their pathogenic ability.The shape,length,composition,and mode of movement of bacterial flagella can all affect the motility of bacteria.These different types of flagella play important roles in bacterial growth,metabolism,chemotaxis,infection,survival,and adaptation to the environment.Therefore,exploring the structure and function of flagella is crucial for deeply understanding the biological characteristics,pathogenic mechanisms,and prevention of bacteria.The structure of flagella is highly conserved among different species and genera.Salmonella flagella is assembled by regulating and secreting substrates required at different stages through the flagella type III secretion system.Flagellar hook associated protein 3（HAP3,FlgL）and flagellar hook associated protein 2（HAP2,FliD）are the constituent proteins of Salmonella flagella,playing an important role in the assembly process of flagella.FliD protein ensures the correct assembly of flagella and enhances the stability of flagella structure through its interaction with FlgL protein.Although the structures of many strains of FliD and FlgL proteins have been analyzed,the structure of the FlgL-FliD complex has not been characterized yet,and the way it interacts is unknown.The mechanism of flagella elongation is still unclear.In this study,the flagellar component proteins FlgL and FliD of Salmonella typhimurium were used as the research objects,and the target protein was successfully expressed in E.coli by constructing the expression plasmid and optimizing the protein expression conditions.High purity HT FlgL_40-286and FliD protein samples were obtained by nickel column affinity chromatography,gel filtration chromatography and other purification methods.The strong interaction between HT-FlgL_40-286and FliD was verified through molecular sieve co elution and Pull Down experiments.The composite of HT-FlgL_40-286and FliD was successfully constructed through molecular sieve co elution and cross-linking,and the composite structure with a resolution of 4.05（?）was analyzed using the cryo-electron microscopy.On the basis of structure,the interaction interface between HT-FlgL_40-286and FliD protein was determined.Mutations were performed at potential interaction sites,and seven mutation combinations affecting Salmonella motility were screened through gene knockout and motility experiments.It was speculated that 266 aspartic acid may have a significant impact on flagella assembly.Different mutations were performed at this site,and it was found that after the 266 amino acid mutation,it will affect the interaction between HT-FlgL_40-286and FliD protein.This study analyzed the structure of the flagellar hook-filament connection protein FlgL and the flagellar cap protein FliD complex in S.typhimurium,determined the interface of interaction between FlgL and FliD,and verified that changes in the interaction between FlgL and FliD affect the motility of bacteria.The research in this study provides a theoretical and structural basis for understanding the mechanism of flagellar elongation and provides ideas for clarifying the mechanism of flagellar filament elongation mediated by FliD in the future.