Final Report Summary - FUTURE T3SS (Bacterial effector secretion: Function and Architecture of the Type 3 Secretion System) A major interest of our group is to decipher general mechanisms and structural aspects of bacterial secretion systems from human pathogens. We focus on understanding how these macromolecular complexes assemble and work to facilitate bacterial infection. Water-borne bacterial pathogens including Salmonella typhimurium and Shigella flexneri utilize a nanosyringe-like structure named Type 3 Secretion System (T3SS) to hijack human cells and prepare them for subsequent invasion. We study the structure and function of the T3SS and take a closer look at the atomic architecture of the T3SS throughout the infection process.In this study, we have attempted to address several structural and functional aspects of effector molecule transport through the T3SS. Therefore, we focused primarily on the molecular transport mechanisms of this conserved bacterial virulence factor. We integrated different interdisciplinary approaches such as cellular microbiology, computational modeling and structural biology methodologies to advance our understanding of the function and assembly of macromolecular structures at highest possible resolution and to determine which approaches can also be applied to other biological pathways beyond infectious diseases.Major questions addressed in this proposal focus on the T3SS structural changes coupled to effector transport and how these macromolecular complexes efficiently facilitate the effector translocation across biological membranes. Therefore we expanded our previous studies on the Salmonella typhimurium needle protein and analyzed othologous needle structures from Shigella flexneri using solid state NMR in combination with genetic and biochemical assays. Our studies revealed the presence of a water filled T3SS transport channel with conserved architecture which is formed by multiple copies of the needle protomer. We also collected conducted cryo-electron microscopy studies of T3SS particles isolated from Shigella flexneri. The structural analysis revealed the 3-dimensional architecture of the Shigella needle complex at near atomic resolution showing novel structural details in the substrate free and the substrate bound Shigella T3SS. Comparison of different transport intermediates at near atomic resolution provided deep insights in structure-function relationship of this secretion systems essential for bacterial virulence of many different Gram-negative bacteria. For these studies we established a novel approach to block the protein transport through the T3SS fusing effector molecules with knot proteins. Furthermore, we analyzed the architecture and the inner surface of the T3SS needle from both Salmonella and Shigella. We identified specific lumen facing residues which play an important role in the substrate transport through the channel. Combining structural information obtained from the Shigella needle, in silico modeling approaches and use of different transport assays allowed us to propose a transport mechanism of effector transport through the T3SS needle.Effector molecule targeting during T3S was another research focus of this study. To decipher how the sorting platform contributes to effector molecule delivery to the T3SS needle complex we performed a broad analysis of the function and architecture of these cytosolic bacterial complex. Our studies revealed the formation of a hetero-oligomer made of 4 different proteins which assembles in higher oligomers in the presence of the needle complex. We analyzed the subunit stoichiometry, the structural organization and the assembly of the sorting platform using biochemical and genetic approaches in combination with different biophysical techniques (native MS, SAXS, X-ray crystallography, MALS SPR)(manuscript under review). Regarding the instantaneous host-pathogen interaction and the role of the T3SS in this process we were interested in the structure-function analysis of the needle tip complex and in understanding how posttranslational modifications of T3SS subunits influence effector secretion. Therefore, we solved the X-ray crystal structures of proteins complexes constituting the T3SS needle tip which might be involved in controlling the release of effector molecules from pathogenic bacteria. In order to understand better the regulation of T3SS we screened for novel proteins which might act in signaling processes upstream of the secretion system complex. In these studies we identified several uncharacterized proteins which might play a crucial role in T3SS regulation.
