A trefoil knot mimic and multiple pore states define the mechanism of a split-shell Tc toxin

Toxin complex (Tc) toxins are among the largest virulence factors secreted by pathogenic bacteria, operating via a sophisticated injection mechanism that translocates cytotoxic effectors into host cells. While most structural and mechanistic studies have focused on fused-shell Tc toxins such as PTC3 from Photorhabdus luminescens , the structurally distinct split-shell toxins remain poorly understood. Here we report cryoelectron microscopy structures of the split-shell Yersinia entomophaga Tc holotoxin (YenTc) in multiple conformational states at resolutions of 2.6 to 3.7 Å. We show that the chitinase subunit YenChi2 is essential for shell expression and stabilizes the N terminus of the shell subunit YenA1 in the prepore state. Our structures also reveal the first trefoil knot mimic in Tc toxins, which serves the dual function of stabilizing the linker domain and holding the two halves of the split shell together. We further describe three distinct pore states: the conventional pore state, an inverted pore state in which the shell rotates 180° around a hinge in YenA2, and a lollipop pore state induced at alkaline pH in which the YenA1-YenChi2 subcomplex fully dissociates from the channel. Strikingly, all three pore states also manifest in the TcA pentamer of P. luminescens PTC3, indicating that they represent a general but previously undescribed feature of Tc toxin pore formation. Taken together, our results establish a mechanistic framework for split-shell Tc toxins and expose a dynamic, multilevel intoxication pathway that may apply broadly to Tc toxins.