Cross-family small GTPase ubiquitination by the intracellular pathogen Legionella pneumophila

Abstract

The intracellular bacterial pathogen Legionella pneumophila ( L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.’s ∼330 secreted effector proteins are ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p hijacks host cell ubiquitin signaling, we generated a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection increases ubiquitination of host regulators of subcellular trafficking and membrane dynamics, most notably ∼40% of mammalian Ras superfamily small GTPases. We determine that these small GTPases undergo non-degradative ubiquitination at the Legionella-containing vacuole membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central role in cross-family small GTPase ubiquitination, and that these effectors function upstream of SidE-family ligases in the poly-ubiquitination and retention of GTPases in the LCV membrane. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. Our findings position L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.

Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

“…Despite their identification, the targets and functions of most of these L.p. effectors are currently unknown 4 . For the effectors whose functions are known, L.p. has been shown to target host proteins that are evolutionarily conserved from amoeba to humans, many of which are key players in regulating membrane traffic in cells 5–9 . Studying the function of L.p. effectors has often led to a deeper understanding of the role of essential host proteins, including a novel posttranslational modification 5 and non-canonical ubiquitination signaling 10–14 .…”