Canonical and phosphoribosyl ubiquitination coordinate to stabilize a proteinaceous structure surrounding the Legionella -containing vacuole

ABSTRACT Legionella pneumophila (L.p.), an intracellular bacterial pathogen, hijacks the ubiquitin signaling network of its eukaryotic host cells to establish infection. Two families of L.p. secreted ubiquitin ligases are instrumental in the maturation of the Legionella-containing vacuole (LCV): the SidC/SdcA family, which catalyzes canonical ubiquitination, and the SidE family, which bypasses the E1-E2-E3 enzymatic cascade and directly conjugates ubiquitin to a target through a phosphoribosyl (PR) linkage. Here, we demonstrate that the coordinated activities of these two effector families generate a hyperstable, ubiquitin-rich structure surrounding the LCV. We propose a model in which an initial wave of SidC/SdcA-mediated canonical ubiquitination around the LCV is further modified by SidE family-driven PR-ubiquitination, resulting in a detergent-resistant “cloud”. The “cloud” is transient, breaking down as infection progresses, suggesting that L.p. reshapes the properties of the proteinaceous shell surrounding the vacuole to meet changing needs throughout its intracellular lifecycle. This unusual structure likely stabilizes and protects the LCV, shielding it from host defense mechanisms during early infection. Our findings reveal cellular consequences of effector interplay during infection and provide a foundation for future studies into the structure and function of the proteinaceous “cloud” surrounding the LCV. Competing Interest Statement The authors have declared no competing interest. Footnotes Additional data has been added regarding the role of the nucleotide binding state of Rab5 in recruitment to the LCV and ubiquitination during infection (see figure S1). The discussion has been expanded to include further detail about the literature regarding Legionella and the endolysosomal system. During data organization, we realized that the representative images for SDS permeabilized WT infected cells for 1- and 2- hours post-infection had been inadvertently swapped in figure 6A. While this does not affect the conclusions of the study, we sincerely apologize for the error, and have corrected it in this revised version. Finally, in recognition of her contributions to the revised version, Puspangana Singh has been added as an author.