Dynamics and activation of the Legionella Type IV secretion system

Abstract

Legionella pneumophila uses the Dot/Icm type IV secretion system (T4SS) to translocate hundreds of effector proteins into host cells, yet how this transenvelope machine assembles and becomes secretion competent remains poorly understood. Here, using cryo-electron tomography and in situ single-particle cryo-electron microscopy, we resolve distinct structural and functional states of the native Dot/Icm T4SS. Unexpectedly, the T4SS predominantly occupies a resting state containing the outer membrane core complex but lacking the inner-membrane complex, including DotA. Recruitment of inner-membrane components establishes a fully assembled but secretion-inactive primed state. A 3.6 [A] cryo-EM structure of the DotO-DotI-DotJ-IcmT complex, integrated with in situ reconstructions, reveals how membrane-associated components organize DotO into a hexamer of dimers ATPase platform. Upon host contact, the T4SS undergoes extensive conformational remodeling while preserving its transenvelope scaffold. Activation involves dilation of the DotG outer membrane gate and transformation of the DotA-IcmX protochannel into an open translocation conduit that extends beyond the bacterial surface. Substrate-associated state exhibit further channel expansion, revealing structural plasticity associated with effector translocation. During macrophage infection, the extracellular conduit bridges the bacterial and host membranes, establishing a direct pathway for effector delivery. Together, our findings reveal a stepwise mechanism of Dot/Icm T4SS channel assembly and activation, in which coordinated remodeling of the secretion channel converts a resting apparatus into a host-engaged translocation machine.