Structural And Functional Insight Into Vibrio Cholerae ⅥSpike Protein VgrG3and Its Cognate Immunity Protein

Bacterial cells have developed multiple strategies to communicate with their surrounding environment. Six different types of protein secretion system, called Type Ⅰ-Type Ⅵ secretion systems (T1SS-T6SS), can be readily identified by their composition and mode of action. T6SS, a large multicomponent and dynamic macromolecular machine, is closely associated with lethal antagonism and has an important role in the interspecies competition of many Gram-negative bacteria. Gram-negative cells that are in close proximity to each other also interact by contact-dependent transport of proteins from a donor cell to a recipient cell via the activity of T6SS apparatus.The T6SS organelle is functionally and structurally analogous to contractile tails of T4bacteriophages. The conserved hemolysin coregulated protein (HCP) that forms a membrane spanning nanotube and VgrG are indispensable for the function of the T6SS machine, and represent two hallmark proteins of T6SS. T6SS is thought to attack cells by initially penetrating them with a trimeric protein complex called the VgrG spike. Neither the exact protein composition of the T6SS organelle nor the mechanisms of effector selection and delivery are clear, but it is very clear that a wide range of toxic effector molecules are the key of T6SS mediated competition, allowing predatory cells to kill prey cells. The donor cells use T6SS to inject toxic effectors into receptor cells and confer fitness advantages on the donor cells. The corresponding immunity proteins are produced by the donor cells to protect themselves against the effector proteins, thereby preventing their self-intoxication. Recently, the additional C-terminal domain of VgrG3in the Vibrio cholerae was identified as a T6SS effector that acts to degrade peptidoglycan, and its immunity protein was determined as TsaB. Information on the molecular mechanism of VgrG3and its immunity protein TsaB has been lacking.Here, we report the crystal structures of the VgrG3C-TsaB complex and native TsaB, and combine with supplemental biochemical experiments to explore the function of VgrG3C and TsaB. VgrG3C-TsaB complex is heterotetramer in solution. VgrG3C displays a phage-T4-lysozyme-type fold. Glu827and Asp842, two invariant amino acids constitute the active dyad residues of VgrG3C. Compared with its structural homologs, VgrG3C displays several unique features, which are associated with its substrate specificity. The immunity protein TsaB interacts with VgrG3C through molecular mimicry, and inserts into the VgrG3C active pocket. Taken together, these data establish a novel inhibitory mechanism and provide the basis for the development of potential antibacterial agents. Importantly, the oligomerization of TsaB is involved in the inhibition of VgrG3C.The dynamic TsaB proteins can effectively bind to the VgrG3C protein in multiple directions. Our work also extends our understanding of the role of T6SS in interspecies competition.