The Legionella pneumophila Dot/Icm T4SS injects ∼ 300
protein effector
proteins into host cells. Dot/Icm substrates have been proposed to contain a carboxy-terminal
signal sequence that is necessary and sufficient for export, although both traits have been demonstrated for only a small fraction of these
proteins. In this study, we discovered that export of the substrate SidJ is mediated by dual
signal sequences that include a conventional C-terminal domain and a novel internal motif. The C-terminal
signal sequence facilitates secretion of SidJ into host cells at early points of
infection, whereas the internal
signal sequence mediates secretion at later time points. Interestingly, only the internal
signal sequence is necessary for complementation of the intracellular growth defect of a ΔsidJ mutant. Although this is the first report of a Dot/Icm substrate being secreted by an internal
signal sequence, many other substrates may be exported in a similar manner. In addition, efficient translocation of SidJ is dependent on the chaperone-like type IV adaptors IcmS/IcmW. Five IcmS/IcmW binding domains that are distinct from both
signal sequences were elucidated and, interestingly, only secretion mediated by the internal
signal sequence requires IcmS/IcmW. Thus, Legionella employs multiple sophisticated molecular mechanisms to regulate the export of SidJ.