Deciphering Legionella effector delivery by Icm/Dot secretion system reveals a new role for c-di-GMP signaling

[Display omitted] •The dynamics of multi-effector secretion is addressed in the paradigm Legionella Icm/Dot T4SS.•A kinetic translocation assay allowing a fine monitoring of the T4SS activity is set up.•Specific secretion patterns consistent with sequential functions in the host are reported.•T4SS-dependent translocation is promoted by a diguanylate cyclase.•Unreported control of Type 4 secretion by the second messenger c-di-GMP signaling is revealed. Secretion of bacterial effector proteins into host cells plays a key role in bacterial virulence. Yet, the dynamics of the secretion systems activity remains poorly understood, especially when machineries deal with the export of numerous effectors. We address the question of multi-effector secretion by focusing on the Legionella pneumophila Icm/Dot T4SS that translocates a record number of 300 effectors. We set up a kinetic translocation assay, based on the β-lactamase translocation reporter system combined with the effect of the protonophore CCCP. When used for translocation analysis of Icm/Dot substrates constitutively produced by L. pneumophila, this assay allows a fine monitoring of the secretion activity of the T4SS, independently of the expression control of the effectors. We observed that effectors are translocated with a specific timing, suggesting a control of their docking/translocation by the T4SS. Their delivery is accurately organized to allow effective manipulation of the host cell, as exemplified by the sequential translocation of effectors targeting Rab1, namely SidM/DrrA, LidA, LepB. Remarkably, the timed delivery of effectors does not depend only on their interaction with chaperone proteins but implies cyclic-di-GMP signaling, as the diguanylate cyclase Lpl0780/Lpp0809, contributes to the timing of translocation.