Phosphoribosyl-linked serine ubiquitination of USP14 by the SidE family effectors of Legionella excludes p62 from the bacterial phagosome

Xenophagy is an evolutionarily conserved host defensive mechanism to eliminate invading microorganisms through autophagic machinery. The intracellular bacterial pathogen Legionella pneumophila can avoid clearance by the xenophagy pathway via the actions of multiple Dot/Icm effector proteins. Previous studies have shown that p62, an adaptor protein involved in xenophagy signaling, is excluded from Legionella-containing vacuoles (LCVs). Such defects are attributed to the multifunctional SidE family effectors (SidEs) that exhibit classic deubiquitinase (DUB) and phosphoribosyl ubiquitination (PR-ubiquitination) activities, yet the mechanism remains elusive. In the present study, we demonstrate that the host DUB USP14 is PR-ubiquitinated by SidEs at multiple serine residues, which impairs its DUB activity and its interactions with p62. The exclusion of p62 from the bacterial phagosome requires the ubiquitin ligase but not the DUB activity of SidEs. These results reveal that PR-ubiquitination of USP14 by SidEs contributes to the evasion of xenophagic clearance by L. pneumophila. [Display omitted] •USP14 is PR-ubiquitinated by Legionella SidEs at multiple serine residues•PR-ubiquitination of USP14 disrupts its interaction with the xenophagy adaptor p62•SidEs-catalyzed modification of USP14 excludes p62 from the bacterial phagosome•SidEs mediate evasion of host xenophagy response in a co-infection system Legionella pneumophila SidE family effectors catalyze phosphoribosyl-linked serine ubiquitination. Ge et al. demonstrate that USP14 is a bona fide PR-ubiquitination target of SidEs, thereby impairing its interaction with the xenophagy adaptor p62. PR-ubiquitination of USP14 inhibits p62 recruitment to the bacterial phagosome, thus contributing to the evasion of host xenophagy.