A virulence strategy used by the intracellular pathogen Legionella pneumophila is to manipulate host cellular processes in order to survive within phagocytic host cells. More than 300 virulent effector proteins are secreted into host cells by the specialized Dot/Icm type IV secretion system (T4SS). Many effector proteins contain hydrophobic transmembrane domains (TMDs) to fulfil their function in host cell membranes. However, the mechanisms T4-secreted TMD effectors (TMEs) use to target and insert into the correct membranes of eukaryotic hosts remain to be elucidated.

To understand the relevance of host cell targeting factors and receptors involved in membrane insertion, the subcellular localization and interacting proteome of bacterial TMEs were assessed either after T4SS-assisted injection or in-host ribosomal translation. The intracellular environment of four Legionella TMEs, differing in hydrophobicity and position of their TMD, was characterized using TurboID-mediated proximity dependent biotinylation. The biotinylated proteins were then captured on streptavidin beads and identified by mass spectrometry.

Proteomic analysis revealed that bacterial TMEs interact with host protein components involved in vesicle trafficking pathways between the endoplasmic reticulum (ER) and the Golgi apparatus, endosomal vesicle transport, protein folding and turnover, and mitochondrial energy metabolism. Moreover, T4-secreted and in-host ribosomal translated TMEs with their TMD located towards the C-terminus could be associated with several proteins involved in the post-translational signal recognition particle (SRP)-dependent ER targeting pathway. To investigate the mechanisms of TME integration at the ER, co-immunoprecipitation and inhibitors that specifically target post-translational pathways will be used. Additionally, fluorescence microscopy will be employed using self-labelling tags to track the route of TMEs in living cells.

These primary findings suggest that T4-secreted TMEs, depending on the position and hydrophobicity of their TMD, use eukaryotic membrane protein biogenesis pathways into the ER and are then trafficked to their specific compartment in the host.