The SidC and SidE families of Legionella pneumophila effectors differentially regulate ubiquitination, morphology, and stable capture of Rab5A at the bacterial vacuole surface

Intracellular bacterial pathogens have evolved to survive in a distinctly hostile environment. Intravacuolar pathogens such as Legionella pneumophila (L.p.) reside within a membrane bound compartment in their host cell, reshaping host materials into a new, protected organelle. The molecular mechanisms employed by L.p. during early infection to defend its vacuole are not entirely understood. As host cells uptake the bacterium by phagocytosis, L.p. must avoid classical trafficking of its phagosome through the endolysosomal pathway or attack by autophagy, which would ultimately lead to destruction of the bacterium in the lysosome. In this work, we set out to define how L.p. manipulates host regulatory proteins to defend its vacuole, termed the Legionella-containing vacuole (LCV), from these threats during early infection. First, we explore the interaction of Rab5, the master regulator of the early endosomal compartment which is required for phagosome maturation, with the LCV. We determine that Rab5 associates with the LCV and is both mono- and polyubiquitinated during infection. This ubiquitination is dependent on two families of secreted bacterial proteins, SidC/SdcA and the SidE family of effectors. Finally, we find that SidE family dependent polyubiquitination is necessary for retention of Rab5 at the LCV membrane. Next, we recognized the unique opportunity presented by Rab5, as this host protein localizes to both the WT and avirulent strain LCV. We determine that Rab5 associated with the WT LCV has a distinctive morphology and stability that is dependent on the SidE family of effectors rather than host regulators. Finally, we discover that L.p. infection induces a burst of ubiquitination of both host and bacterial proteins, and that SidC/SdcA play a central role in this burst. We propose a new role of SidC/SdcA as a metaeffector, which regulates the activity of other as yet unknown bacterial proteins. Taken together, our findings suggest a model in which ubiquitin