Resolving Legionella's Dot/Icm machine in situ at near-atomic resolution, this work identifies DotA–IcmX as the activation-gated secretion conduit, overturning prior models and explaining how a major pathogen deploys 330+ effectors across bacterial and host membranes.
The Legionella Dot/Icm type IV secretion system employs a pentameric DotA-IcmX protochannel as a gatekeeper that maintains the system in an inactive state through a narrow hydrophobic lumen, and upon host contact undergoes extensive conformational rearrangements to form an extended transenvelope channel that enables direct translocation of effector proteins from the bacterial cytoplasm into host cells.
The Dot/Icm machine in Legionella pneumophila is one of the most versatile type IV secretion systems (T4SSs), with a remarkable capacity to translocate over 330 different effector proteins across the bacterial envelope into host cells. At least 27 Dot and Icm proteins are required for assembly and function of the system, yet the architecture and activation mechanism remain poorly understood at the molecular level. Here, we deploy cryo-electron microscopy to reveal in-situ structures of the Dot/Icm machine at near-atomic resolution. Importantly, two proteins essential for effector translocation, DotA and IcmX, form a pentameric protochannel at an inactive state. Upon activation, the DotA-IcmX protochannel undergoes extensive rearrangements to form an extended transenvelope passage capable of transporting effector proteins from the bacterial cytoplasm into host cells as revealed by cryo-electron tomography. Collectively, our findings suggest that the DotA-IcmX complex functions as the gatekeeper for effector translocation of the Dot/Icm T4SS.