A multidisciplinary and integrative approach for deciphering the assembly, structure and dynamics of a contractile tail baseplate – T6-PLATFORM

Contractile injection machines are fascinating specialized nano-structures evolved by bacterial and viral pathogens to deliver macromolecules into target cells. These machines have been elaborated for different purposes such as the injection of DNA into host cells by bacteriophages or for the delivery of toxin effectors into target cells by R-pyocins or Type VI secretion systems (T6SS). We have shown that the T6SS is involved in bacterial competition by delivering toxins into competitor bacteria, and therefore this apparatus provides an advantage for the bacterium in its environment. At the molecular level, contractile tails comprise an inner needle (or inner tube) wrapped by a sheath, built on an assembly platform - the baseplate. The sheath is assembled in an extended conformation that stores the mechanical energy necessary for its contraction and to propel the inner tube towards the target. The baseplate controls the assembly of the tube/sheath structure, its contraction and the selection/sorting of toxins. In addition, we recently reported that the T6SS baseplate docks onto a trans-envelope complex that positions and orients the tail toward the target cell and serves as a channel for the passage of the needle during sheath contraction. We recently identified the components of the T6SS baseplate. It comprises the VgrG, TssE, TssF, TssG and TssK proteins. In addition, we found that the TssA protein associates first with the trans-envelope complex, recruits and stabilizes the baseplate and coordinates the extension of the tube with that of the sheath. Our current work aims to gain insights onto the assembly and structure of the baseplate as well as understanding the detailed role of TssA during T6SS biogenesis. Here, we will purify the T6SS baseplate as well as intermediate complexes. To obtain structural information, all these complexes will be (i) imaged by negative-stain and cryo-electron microscopy as well as by cryo-electron tomography to obtain structural information, (ii) analysed by chemical cross-linking combined to mass spectrometry and native mass spectrometry to gain details on the subunit contacts and the complex topology, and (iii) modeled through an integrative approach that determines the architecture of their constituents. Finally, TssA interactions during T6SS biogenesis will be defined by fusing TssA to the biotin ligase and by identifying biotinylated partners. Such a project requires the development of state-of-the-art techniques, such as high-resolution electron microscopy, cryo-electron tomography, mass spectrometry, in vivo reporter assays, and novel computational methodologies. We have therefore constituted a network of collaborations with internationally recognized teams to develop these techniques with the goal to provide an unprecedented knowledge on the T6SS. The project combines an interdisciplinary consortium with expertise on bacterial genetics, biochemistry, mass spectrometry, fluorescence and electron microscopy and tomography. The multi-scale information gathered will be employed to build models on the assembly, structure and dynamics of the T6SS.