Identification And Functional Analyses Of T3SS-related Protein-protein Interactions In Yersinia Pseudotuberculosis

Type III secretion system(T3SS) is an important virulence system in Gram-negative pathogenic bacteria. The Ysc-Yop T3 SS of Yersinia is encoded by a virulence plasmid called p YV or p CD and translocated Yops effector proteins into cytosol of host cells by a needle injection structure to achieve the purpose of pathogenic. The assembly of Yersinia Ysc-Yop T3 SS apparatus and secretion of Yops are two very complex processes that require the participation of many factors, and protein-protein interactions play important roles in these processes, which are not clearly understood.In this study, bacterial two-hybrid system was first used to comprehensively screen T3 SS associated interaction proteins encoded by the p YV virulence plasmid in Yersinia pseudotuberculosis strain YPIII. Through literature survey, possible novel protein-protein interactions that may contribute to the understanding of T3 SS structure assembly as well as Yops secretions were selected for further studies. This thesis is comprised of the following three chapters:1. Protein-protein interactions of Yersinia T3 SS components by bacterial two-hybrid analysis. A total of 43 protein-protein interactions were identified by Blue-White selection followed by β-galactosidase activity assay, among which 22 have previously been reported. Of the rest interaction proteins, 12 were self-interacting proteins, indicating these proteins may form multimers in the physiological process. Ysc Q and Ysc O have been reported to play indispensable roles in T3 SS, but the exact mechanism is unclear. Meanwhile, the secretion processes of Yop H and Yop M are unclear. Ysc Q-Yop H and Ysc O-Yop M interactions were choosen to further explore the roles of Ysc Q and Ysc O during the secretion process of Yop H and Yop M, respectively.2. Confirmation of protein-protein interactions and analyses the interaction regions of Yop H-Ysc Q and Yop M-Ysc O. GST pull-down assay was used to validate the interactions of Yop H-Ysc Q and Yop M-Ysc O. The tertiary structures of Yop H,Ysc Q, Yop M and Ysc O were first analysised. By truncation of these proteins and followed by bacterial two-hybrid analyses, several interacting regions were identified.Yop HN-140 aa, which plays an important role for secretion of Yop H, was confirmed to interact with Ysc Q218aa-C. ysc Q encodes full-length of Ysc Q and Ysc Qc, Ysc Qc has the same sequence with Ysc Q218aa-C, indicating that Ysc Q218aa-C and Ysc Qc may play arole in the secretion process of Yop H. Yop MN-100 aa interacts with Ysc O, but the interacting region in Ysc O was not charactertized, which leads us to spetaculate that the interaction between Yop M and Ysc O depends on the integrity of Ysc O.3. Functional analysis of Ysc Q and Ysc Qc. The interaction of Ysc Qc and Yop H has been verified by in vitro pull-down assay. Ysc Q and Ysc Qc are required for T3 SS.To find the interacting sites of Yop H and Ysc Qc as well as to explore the secretion mechanism of Yop H, Ysc Q and Ysc Qc were overexpressed, and a feedback inhibition to Yops secretion was observed, and this inhibition can be relieved by the deletion of lcr Q, which provides information for further in-depth studies of how Ysc Q/Ysc Qc regulate T3 SS and how Lcr Q be secreted to extracellular.