Study On The Signal Transduction Mechanism Of QseEF And Substrate Sorting Mechanism Of T3SS In Salmonella

Salmonella is a common food-borne enteric pathogen that infects both humans and animals.Salmonella infection can cause food poisoning,typhoid fever,and other diseases.In the past few decades,the overuse of antibiotics has promoted drug-resistance development in Salmonella.Therefore,it’ s urgent to develop new anti-infective drugs against Salmonella.The new strategy is to target the key pathogenic elements of bacteria to reduce their infection or virulence,but not to kill them.Thus,the two-component system(TCS)and the type Ⅲ secretion system(T3SS)have become important targets for the development of anti-infective drugs against Salmonella.This study consists of two parts.The first part focuses on the signal transduction mechanism of the two-component system QseEF.QseEF in can sense environmental signal to regulate its GlcN6P synthesis and enhance its virulence.The second part focuses on the mechanisms of how the core components SctV family proteins are involved in T3SS assembly and substrate sorting.The main results of this study are as follows:1.The QseEF complex was successfully obtained by constructing different truncations of QseF and QseE.We observed that the RD domain of QseF interacts with the DHp-CA domain of QseE,and the interaction requires the σ54 interaction domain or the assistance of BeF3-.The mutation of His259 to Glu259 at the site of QseE receiving the phosphate group also facilitates the interaction between the QseE and QseF.2.Due to their unique properties,the expression level of membrane proteins is often quite low in vitro,such as the SctV protein.This problem was solved by constructing the chimeric protein ISS(InvATM-SsaVL-SsaVc).Given the strong hydrophobicity of the transmembrane region,it’s essential to select suitable detergents to stabilize the conformation of membrane proteins.After screening a series of detergents,we found that GDN could help to obtain a stable chimeric protein ISS with a conformation that is similar to the one under natural physiological conditions.This is essential for further three-dimensional structure analysis by Cryo-EM.3.The SpiC-SsaM/SsaL is a chaperone/gatekeeper complex of T3SS in Salmonella Typhimurium SPI-2.We obtained this complex by bacterial co-expression.Limited proteolysis assay on this complex indicated that the N-terminal loop region of SsaL is the critical interface of its interaction with the chaperone SpiC-SsaM.4.SrcA is a multi-cargo chaperone that binds various T3SS-secreted effectors in Salmonella SPI-2.In this study,the potential SrcA-binding effectors were screened by a bacterial co-expression system.The results show that SrcA only interacts with the effector protein SteD,but not SseL,PipB2 or SseK2.This work not only helps to understand the mechanisms of QseEF signal transduction and the SctV-involved secretion process of T3SS,but also provides theoretical support for the development of new anti-infective drugs against Salmonella.