Intracellular pathogens manipulate host organelles to support replication within cells. In the case of Legionella pneumophila, the bacterium translocates proteins that establish an endoplasmic reticulum (ER)-associated replication compartment. Using high-resolution electron microscopy and fluorescence microscopy we show that the bacterial Sde proteins target host reticulon 4 (Rtn4) to control tubular ER dynamics, resulting in tubule rearrangements as well as structural alterations to Rtn4 proximal to the Legionella pneumophila containing vacuole (LCV). We utilized both in vitro recombinant protein systems and L. pneumophila infection models to characterize the Sde proteins we were able to identified the molecular mechanism promoting these ER rearrangements. Sde ubiquitin (Ub) ligation to Rtn4, which occurs almost immediately after bacterial uptake, is dependent on two sequential enzymatic activities expressed from a single Sde polypeptide: an ADP-ribosyltransferase (ART) and a nucleotidase/phosphohydrolase (NP). The ADP-ribosylated moiety of ubiquitin is a substrate for the nucleotidase/phosphohydrolase, resulting in either transfer of ubiquitin to Rtn4, or phosphoribosylation of ubiquitin in the absence of a ubiquitination target. These enzymatic activities collaborate to promote ubiquitin ligation through a phosphodiester bond, rather than the classic isopeptide ubiquitin linkage and specifically target Rtn4 and a subset of Rabs, including Rab33b and Rab1.

These enzymatic activities are essential for intracellular replication and ER association in amoeba, but not macrophages. We show Sde targeting of LCV associated polyUb conjugates with ADP-ribosylate or phosphoribosylate modifications protect these products from host disassembly and are required for intracellular replication in amoeba. We also identified a third enzymatic domain in Sde proteins, a K63-polyubiquitin specific deubiquitinase (DUB) that limits LCV polyUB within the first 30 minutes of infection that fuels Sde Ub ligase function by generation of a free monoubiquitin substrate pool, while also preventing lysosomal targeting ubiquitin signals (K63) from accumulating on the Legionella vacuole membrane. This work demonstrates that Sde mediated ubiquitination transforms peripheral ER tubules associated with the LCV through a novel molecular mechanism, where a single bacterial protein catalyzes a multistep biochemical pathway to control ubiquitination via both ubiquitin conjugation and deconjugation.