Structural And Functional Studies On Pseudomonas Aeruginosa T6SS Effector Tse3and Human Innate Immunity Protein STING

Gram negative bacteria using a syringe-like tool of Type VI secretion system (T6SS) inject the effector proteins into the rival cells in a cell-contact manner leading to their death. Concurrently, bacteria employ cognate immunity proteins neutralizing these effector proteins to prevent self-intoxition. This effector-immunity pairs (E-I pairs) of T6SS endow the bacteria with great advantage in the fierce niche competiton. Three effector proteins (Tsel, Tse2and Tse3) controlled by H1-T6SS from Pseudomonas aeruginosa have been identified by Secretome and Mass spectrum analysis. Tsel and Tse3were demonstrated to be the amidase and muramidase degrading the peptide and glycan moiety of peptidoglycan (PG) respectively. In this thesis, X-ray crystallography method was performed to elucidate the mechanism used by Tse3and Tsi3in details. Tse3has an annexin repeat-like fold at the N-terminus and a G-type lysozyme fold at the C-terminus. Interestingly, one loop (loop12) at the N-terminal domain, together with one helix (a9) from the C-terminal domain, anchors Tse3or the Tse3-Tsi3complex to membrane in a calcium-dependent manner in vitro. Moreover, the membrane-binding ability is shown to be essential for Tse3’s activity. In the C-terminal domain, a Y-shaped groove occurs on the surface probably serving as the PG-binding site. Two calcium-binding motifs are also observed in the groove and they are necessary for Tse3’s activity. In the Tse3-Tsi3structure, three loops of Tsi3insert into the substrate-binding groove of Tse3. Three calcium ions found in the interface of the complex are demonstrated to be indispensable for the formation of Tse3-Tsi3complex. These findings further our perspective on the T6SS effector proteins and provide the structural basis for eliminating and combating the pathogenic infection.STING protein plays an essential role in the double strands DNA sensing pathway in the cytosol for innate immunity. The DNA sensors relayed the signal to STING which recruited the kinase TBK1and transcript factor IRF3. Then, IRF3was phosphated by TBK1, which leaded to its dimerization. Dimerized IRF3was transferred to the nucleus and promote the transcription of type I INF which initialized the anti-pathogen process by innate immunity. Recently, the cyclic dinucleotides (CDNs) pathway was discovered making adaptor protein STING more fascinating. In this thesis, we solved the crystal structures of h-STINGCTD domain and its complex with c-di-GMP. The h-STINGCTD and c-di-GMP-h-STINGCTD complex behaves as a dimmer in the solution and crystal indicating it may function as a dimmer. The c-di-GMP was found in the dimmer interface of two STING protomer with a moderate binding affinity. Comparing to the apo form structure, c-di-GMP binding did not induce significant conformation change occurring in h-STINGCTD. However, the metazoan second messenger2’5’-cGAMP binded to h-STINGCTD resulting in drastic structural changes:The open state h-STINGCTD became close state; Loops between β2and β3became anti-parallel β sheet capping the CDNs in the pocket formed by two STING protomers. At the same time, the c-di-GMP did not induce significant immune response mediated by h-STING in vivo. Considering the complicated results of type I INF for the infection of bacteria, they may hijack the CDNs pathway for their purpose by mimcing the metazoan second messenger and human STING protein may evade this bacterial strategy in the evolution process. As for the mouse STING protein, it always adopts the close form and strongly responses to stimulates of CDNs, which indicates the functional differences of STING protein among species. All in all, our structural information of h-STINGCTD provides the molecular basis for the therapy of infection and autoimmune diseases such as systemic lupus erythematosus, SLE.