Mechanistic and structural insights into the type VI secretion system tail of "vibrio cholerae"

Bacteria use many different types of secretion systems to interact with and modulate their environment. Secretion systems are involved in these tasks with a wide variety of functions. Their substrate proteins mediate motility, phagosomal escape, host modulation or interbacterial competition. Various different mechanisms exist, how the transport across membranes is mediated. Some substrates are secreted in two steps, others in only one step. Some secretion systems just export their cargo into the extracellular medium, whereas others deliver their substrates under specific circumstances into other cells. The Type VI Secretion System (T6SS) is the largest secretion system and can span the entire length of a bacterium. It contracts a long sheath and pushes an inner tube with associated effectors into a target cell. The tail is anchored to the cell envelope by a membrane complex and consists of sheath, tube and baseplate. The baseplate initiates polymerization of the sheath-tube complex and connects the sheath to the membrane complex. The membrane complex is formed by the membrane proteins TssJ, TssL and TssM. TssJ is located in the outer membrane, whereas TssL and TssM are inner membrane proteins. The baseplate is formed by TssE, TssF, TssG and TssK. TssK connects the baseplate to the membrane complex. TssF and TssG form the baseplate wedge and TssE connects the sheath to the baseplate. The central part of the baseplate consists of VgrG and PAAR-proteins. A trimer of VgrG-proteins forms the spike and the monomeric Zinc-containing PAAR-protein forms the spike tip. The inner Hcp-tube polymerizes on VgrG and the sheath polymerizes on TssE and wraps around the Hcp-tube. Polymerization of the sheath is mediated by a TssA cap at the growing end of the sheath, opposite of the baseplate. The sheath is a six-start helix of TssB and TssC (VipA and VipB) and its protomers form an interlaced network that is essential for stability of the sheath. Presumably, the baseplate triggers contraction of the sheath, which leads to an increase in the helical twist, widening of the diameter and shortening of the length of the sheath. The contraction pushes the inner tube with the associated spike and effectors into target cells. The contracted sheath is specifically recognized by the AAA+-protein ClpV that disassembles it. In recent years our understanding regarding structure and function of the T6SS advanced rapidly. However, certain aspects are not yet understood. This thesis will address progress on understanding different aspects of the T6SS mostly from a structural and mechanistic perspective. First, I will briefly describe other bacterial secretion systems and then present our findings on (i) the structure of the T6SS sheath in a contracted state, (ii) the mechanism of contraction of the sheath, (iii) the structure of the sheath in an extended state and (iv) structures of associated components, namely the baseplate and the cap. We solved the structure of the T6SS-sheath in a contracted state, discovered that it forms an interlaced helix and that the interlacing linkers are important for its function. We also proposed how specificity of ClpV binding to the contracted sheath is achieved. Further targeted mutagenesis lead to a model of how sheath contraction is propagated through the sheath. Some mutations stabilized the sheath in an extended state and made it possible to isolate it. Extended sheaths contain the inner Hcp-tube.We then structurally characterized the extended sheath-tube assembly and gained further insights into the rearrangement of domains as well as a deeper understanding of the binding mode of ClpV. The extended sheaths were also specifically associated with proteins of the baseplate and the cap. We described the structural arrangement of baseplate proteins and the cap and gained first insights in the relative orientation with the sheath in a near-native assembly. I will provide insights into the complete assembly of the cytosolic part of the T6SS. The knowledge about the T6SS that is presented here, can likely be transferred to related systems as contractile tailed phages or R-type pyocins.