Type IV secretion systems (T4SSs) are large macromolecular complexes that transport proteins and nucleic acids across bacterial membranes. They play essential roles in pathogenesis, yet the mechanism of cargo translocation remains unresolved. Here, we used a host-adhesion-without-internalisation (HAWI) system to induce T4SS effector secretion in Legionella pneumophila while preventing bacterial entry into the host, enabling direct visualisation of the active T4SS. A combination of in situ cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) revealed structural rearrangements of the T4SS during active translocation. The L. pneumophila T4SS is organised as a stacked assembly of five subcomplexes: the outer membrane complex (OMC); the periplasmic ring (PR), positioned immediately beneath the OMC; a plug located in the lumen of the PR; a channel linking the PR to the inner membrane; and a cytoplasmic ATPase complex.
In the active T4SS, the OMC tilts ~10° towards the inner membrane and remodels at its centre to form an ~4 nm pore in the outer membrane. The PR contracts laterally from 10 to 5 nm, coupled to displacement of the central plug density. Conversely, the channel widens axially to create an ~4 nm conduit from the inner membrane to the PR. Together, these changes generate an unobstructed passage from the inner membrane, through the periplasm, to the extracellular space, providing the first structural insights into the reorganisation required for effector secretion through the L. pneumophila T4SS.
To further probe the translocation mechanism, a poison-domain-containing effector was used to stall secretion and trap intermediates. These “stalled” complexes enabled detailed analysis of the tripartite ATPase assembly (DotO, DotB, DotL) and revealed that DotO, DotB, and the coupling complex adopt a three-tier stacked arrangement. These states provide mechanistic insight into how substrates are recruited and transferred into the secretion conduit. Biochemical analyses further show that the gating proteins DotA and IcmX are secreted yet remain associated, supporting a DotA–IcmX gating mechanism that links OMC/PR remodelling to effector translocation. Overall, these observations provide a mechanistic framework for understanding T4SS activation and effector delivery, and a structural foundation for future therapeutic or biophysical investigations.