Cryo-EM structure of a type VI secretion system-delivered membrane-depolarizing toxin involved in bacterial antagonism.

Many Gram-negative bacteria use type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighboring cells. Proteins in the VasX toxin family form ion-permeable channels in the bacterial cytoplasmic membrane that dissipate the proton motive force, thereby interfering with essential physiological processes. However, the 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 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 molecular insights into the structure and mechanism of VasX family effectors.