Colautti, J.; Shatskiy, D.; Bates, E.; Bullen, N. P.; Belyy, A.; Whitney, J. C.

Many Gram-negative bacteria use type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighboring competitor cells. Members of the VasX protein family, such as VasX from Vibrio cholerae and Tke5 from Pseudomonas putida, disrupt the inner membrane of target cells by forming ion-permeable channels that dissipate the proton motive force, thereby interfering with essential physiological processes. However, the molecular structure of any VasX family effector has remained unknown. Here, we present a cryo-EM structure of Ptx2, a recently identified VasX family effector exported by a T6SS of Pseudomonas aeruginosa. Our structure reveals that Ptx2 is an elongated, multi-domain protein that bears little resemblance to proteins of known function. Notably, the apparent flexibility of its domains suggests that Ptx2, like other membrane-depolarizing toxins, undergoes substantial conformational changes to facilitate membrane insertion. Guided by these predicted structural rearrangements, we used mutagenesis coupled with phenotypic assays to identify key features required for its toxic activity. Together, these findings provide the first molecular level insights into the structure and mechanism of VasX family effectors and expand our understanding of how these proteins contribute to interbacterial antagonism.