Structural Characterization Of The Type Ⅲ Secretion Effector Protein SopB From Salmonella Typhimurium

Food poisoning induced by Salmonella is a global food safety and public health problem.Salmonella can directly inject effector proteins into the eukaryotic host cells through the type Ⅲ secretion system(T3SS),promoting Salmonella invasion and regulating host cell immune responses,leading to disease.Therefore,studying the structure and function of effector proteins can provide a new approach to the development of new drugs,which has important scientific significance and practical application value.Salmonella outer protein B(SopB)is an effector protein secreted by Salmonella through the type Ⅲ secretion system that plays multiple roles during host cell invasion,such as inducing cytoskeletal rearrangement and membrane ruffling,inducing the formation and maturation of salmonella-containing vacuoles(SCV),and activating serine/threonine kinase proteins AKT to inhibit host cell apoptosis.Although the functions of SopB have been reported in many studies,the precise molecular mechanisms by which SopB leads to these effects remain unclear.This study focuses on the phosphoinositide phosphatase SopB from Salmonella Typhimurium.Through the construction of expression plasmids and optimization of protein expression conditions,the target protein was successfully expressed in Escherichia coli.The SopB(29-561)protein sample was obtained by nickel affinity chromatography,gel filtration chromatography,and other purification methods with high purity.Using cryo-electron microscopy,we eventually determined the structure of SopB with a resolution of 2.63 (?).Our results show that SopB is a homo-hexamer formed by the oligomerization of six monomers through electrostatic and hydrophobic interactions.The interaction between SopB and the ATPase Inv C of the type Ⅲ secretion system was validated,and attempts were made to stabilize the complex.However,the dynamic nature of the complex makes it difficult to obtain the structure.Structural analysis revealed that the N-terminal domain of SopB is a conserved small GTPase-binding domain.8 kinds of GTPases in different functional categories were selected to characterize the interaction with SopB using isothermal titration calorimetry(ITC),and three GTPases that interact with SopB were identified: the human GTPase Cdc42 and the bacterial GTPases Mnm E and Rsg A.This study aims to resolve the structure of SopB and investigate its interaction with the cytoplasmic component ATPase Inv C of the type Ⅲ secretion system,as well as screen for GTPases that interact with SopB.These efforts will provide a foundation for further studies on the structure of the effector protein SopB in complex with the cytoplasmic component ATPase Inv C of the type Ⅲ secretion system and with protein targets in eukaryotic cells.