Bacterial Type IV secretion systems (T4SSs) are large macromolecular complexes responsible for transporting proteins across bacterial membranes and facilitating horizontal gene transfer. These dual roles make T4SSs clinically significant, as various bacterial intracellular parasites utilize T4SSs during the infection of human cells. Additionally, T4SS-mediated horizontal gene transfer contributes to the spread of antibiotic resistance genes. Despite intensive research on T4SSs due to their clinical implications, the mechanism by which T4SSs translocate effector proteins or DNA remains elusive in all known organisms.
In this study, we employed a memetic system to trigger T4SS effector protein secretion in the model organism, Legionella pneumophila, a clinically significant intracellular bacterial parasite. The distinctive feature of our approach is its ability to induce T4SS effector protein secretion without necessitating bacterial internalization into the host cytoplasm. This aspect addresses a major challenge previously faced in studying effector protein secretion through the T4SS. To analyse the actively secreting T4SSs, we combined Cryo-Electron Tomography with Sub-Tomogram averaging.
Our memetic system's in-situ T4SS sub-tomogram averages reveal multiple large-scale structural rearrangements. Most notably, the outer membrane core complex (OMCC) exhibits a pronounced 45° shift downwards towards the inner membrane. This downward movement of the OMCC correlates with protein density changes both at the OMCC-inner membrane interface and the OMCC-periplasmic ring (PR) interface. These structural shifts offer preliminary insights into the structural reorganization required for effector protein secretion via the Legionella pneumophila T4SS.